Dry powder composition comprising long-chain rna

ABSTRACT

The present invention is directed to a storage-stable formulation of long-chain RNA. In particular, the invention concerns a dry powder composition comprising a long-chain RNA molecule. The present invention is furthermore directed to methods for preparing a dry powder composition comprising a long-chain RNA molecule by spray-drying. The invention further concerns the use of such a dry powder composition comprising a long-chain RNA molecule in the preparation of pharmaceutical compositions and vaccines, to a method of treating or preventing a disorder or a disease, to first and second medical uses of such a dry powder composition comprising a long-chain RNA molecule and to kits, particularly to kits of parts, comprising such a dry powder composition comprising a long-chain RNA molecule.

The present invention was made with support from the Government underAgreement No. HR0011-11-3-0001 awarded by DARPA. The Government hascertain rights in the invention.

The present invention is directed to a storage-stable formulation oflong-chain RNA. In particular, the invention concerns a dry powdercomposition comprising a long-chain RNA molecule. The present inventionis furthermore directed to methods for preparing a dry powdercomposition comprising a long-chain RNA molecule by spray-drying. Theinvention further concerns the use of such a dry powder compositioncomprising a long-chain RNA molecule in the preparation ofpharmaceutical compositions and vaccines, to a method of treating orpreventing a disorder or a disease, to first and second medical uses ofsuch a dry powder composition comprising a long-chain RNA molecule andto kits, particularly to kits of parts, comprising such a dry powdercomposition comprising a long-chain RNA molecule.

In gene therapy and many other therapeutically relevant biochemical andbiotechnological applications, the use of nucleic acid molecules fortherapeutic and diagnostic purposes is of major importance. For example,rapid progress has occurred in recent years in the field of genetherapy, where promising results have been achieved. Nucleic acids aretherefore regarded as important tools for gene therapy and prophylacticand therapeutic vaccination against infectious and malignant diseases.

Other than DNA, application of RNA also represents a favored tool inmodern molecular medicine. It also exhibits some superior propertiesover DNA cell transfection. As generally known, transfection of DNAmolecules may lead to serious complications. For example, application ofDNA molecules bears the risk that the DNA integrates into the hostgenome. Integration of foreign DNA into the host genome can have aninfluence on the expression of host genes and can trigger the expressionof an oncogene or the inactivation of a tumor suppressor gene.Furthermore, an essential gene—and, as a consequence, the product ofsuch an essential gene—may also be inactivated by the integration of theforeign DNA into the coding region of the gene. The result of such anevent may be particularly dangerous if the DNA is integrated into agene, which is involved in regulation of cell growth. Notwithstandingthe risks associated with its application, DNA still represents animportant tool. However, these risks do not occur if RNA, particularlymRNA, is used instead of DNA. An advantage of using RNA rather than DNAis that no virus-derived promoter element has to be administered in vivoand no integration into the genome may occur. Furthermore, the RNA, inorder to exert its function, does not need to overcome the barrier tothe nucleus.

However, a main disadvantage of the use of RNA is its instability. Eventhough it is understood that DNA, such as naked DNA, when introducedinto a patient circulatory system, is typically not stable and thereforemay have little chance of affecting most disease processes (see e.g.Poxon et al., Pharmaceutical development and Technology, 5(1), 115-122(2000)), the problem of stability becomes even more prominent in thecase of RNA. It is generally known that the physico-chemical stabilityof RNA molecules in solution is extremely low. RNA is susceptible tohydrolysis by ubiquitous ribonucleases or by divalent cations and istypically rapidly degraded, e.g. already after a few hours or days insolution. Rapid degradation occurs even in the absence of RNases, e.g.when RNA is stored in solution at room temperature for a few hours ordays.

To avoid such rapid degradation, RNA (in solution) is typically storedat −20° C. or even −80° C. and under RNAse free conditions to preventdegradation of the RNA. Such storage conditions, however, do notsufficiently prevent a loss of function over time. Additionally,applying such conditions is very cost-intensive, especially for shippingand storage, e.g. whenever such low temperatures have to be guaranteed.

The only method for stabilization of long-chain RNA, which is known andapplied, comprises lyophilization or freeze-drying of the RNA (see e.g.WO2011/069587 and WO2011/069586). Lyophilization is a method, which isknown and recognized in the art to enhance storage stability oftemperature sensitive biomolecules, such as nucleic acids. Duringlyophilization, water is typically removed from a frozen samplecontaining nucleic acids via sublimation. The process of lyophilizationis usually characterized by a primary and a secondary drying step. Inthe primary drying step, free, i.e. unbound, water surrounding thenucleic acid (sequence) and optionally further components, evaporatesfrom the frozen solution. Subsequently, water that is bound by thenucleic acids on a molecular basis may be removed in a secondary dryingstep by adding thermal energy. Thereby, the hydration sphere surroundingthe nucleic acids is lost. Lyophilization is the most common processingmethod for removing moisture from biopharmaceuticals, and it canincrease stability, temperature tolerance, and shelf life of theseproducts. However, lyphilization does have its limitations, especiallyif scale-up is needed. One major disadvantage of lyophilization is thatevery single vial containing a sample has to be lyophilized separately.The lyophilized product cannot be separated into distinct charges oraliquots, as the lyophilized product is not provided e.g. in powderform. Instead, a cake-like product is obtained by lyophilization, whichcannot be divided into distinct charges or aliquots. Therefore, if apowder-like product is desired, a further step of granulation must becarried out. At present, lyophilization of samples for a scaled-upproduction involves cost-intensive equipment, since, for example, a lotof lyophilizers are needed for market production, requiring largeproduction facilities. Together with the time required forlyophilization and the additional requirement of a granulation step thatrenders lyophilization a technique, which is often not suitable forindustrial scale production. Especially in an environment where budgetsare tightening, and where time and facility space are at a premium,lyophilization may not be considered, e.g. by the pharmaceuticalindustry, as a competitive process.

A minor number of case reports refer to spray-dried ribonucleotides.Double-stranded short interfering RNAs (siRNA) for inhalation were driedby using a spray-drying technology. Jensen et al. (2010) studiedparameters to be applied in spray drying of siRNA-loadedpoly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) for providingnano-composite micro-particles for inhalation. As siRNA was believed todenature at temperatures above 55° C., that study applied relatively lowdrying temperatures (T_(inlet)=45° C.; T_(outlet)=30° C.) and highexcipient concentrations (10-30%) to achieve a dry powder with aresidual humidity of less than 4%.

US2011/077284 discloses the provision of dry powders of therapeutic andinhalable short siRNAs against influenza virus. Therein, dryingtemperatures were determined experimentally. Generally, the inlettemperature was from about 65° C. to about 125° C., while the outlettemperature ranged from about 30° C. to about 70° C. In the examplespresented therein, siRNAs are dried at a T_(outlet) of ≤55° C. Thepowders generally had a moisture content of typically less than 10% byweight, or less than 5% by weight, or less than 3% by weight. Also, thestudy characterized the chemical stability of the dry powder. Less than10% by weight of the active siRNA were degraded upon storage of the drypowder composition under ambient conditions for a period of 18 months.However, the biological activity of the siRNA stored as a dry powder wasnot determined.

Summarizing the above, there is a long-lasting and urgent need in theart to provide means, which allow (a skilled person) to store long-chainRNA without loss of activity, an effect, which is commonly observed,particularly in in vivo applications. In this context, a challengingproblem resulting from prior art approaches is to ensure stability ofnucleic acids, particularly stability upon storage and delivery oflonger single-stranded RNA. Another problem of the prior art is the lossof biological activity of nucleic acids subsequent to storage. Finally,by using prior art methods, only small amounts of nucleic acid areobtained. The provision of a suitable form for delivering these nucleicacids but also the production, transport and storage thereof, especiallytransport of RNA, is an issue due to the conditions to ensuretemperatures of −20° C. and less for shipment. Furthermore,lyophilization of long-chain RNA, especially for the use as medicament,bears the problem that it is very cost- and time-intensive, particularlyif commercial production in a scaled-up process is envisaged.

The underlying object is therefore to provide a nucleic acid molecule,in particular a long-chain RNA, exhibiting no loss of activity whenstored prior to its use and being available by cost-avoiding productionprocess. In particular, it is an object of the present invention toprovide a long-chain RNA molecule in a storage-stable formulation. Oneobject of the invention is to provide a dry powder compositioncomprising a long-chain RNA molecule. In addition, it is an objective ofthe invention to provide a method for preparing a dry powder comprisinga long-chain RNA molecule, wherein the RNA molecule retains its chemicalintegrity and its biological activity. It is another object of thepresent invention that such methods are applicable under industriallarge-scale production conditions, preferably by a continuous process.It is a particular object to provide a method that allows drying aliquid comprising long-chain RNA molecules.

The object underlying the present invention is solved by the claimedsubject-matter.

In a first aspect, the invention relates to a long-chain RNA molecule ina particulate formulation. In particular, the invention concerns a drypowder composition comprising a long-chain RNA molecule. Prior to theinvention described herein, long-chain RNA (in contrast to shorterdouble-stranded RNAs) was never provided as a dry powder composition.Advantageously, the dry powder composition according to the inventionprovides a storage-stable form of a long-chain RNA molecule. Inaddition, the dry powder composition according to the invention ischaracterized by superior handling properties. For example, theinventive dry powder composition can be packaged in any quantity or inany container or dosage form, respectively. Handling of the dry powdercomposition according to the invention is further improved by itsfree-flowing properties. In particular, the inventive dry powdercomposition does not form agglomerates or aggregates that would inhibitpackaging and/or dosage. Due to its flowability, the dry powdercomposition according to the invention can be readily further processed.For instance, the dry powder composition can be transferred, e.g. fromone vessel to another or from a larger vessel into a plurality ofsmaller vessels, simply by pouring. The inventive dry powder compositioncan readily be packaged in a variety of packages and final dosage formsaccording to the actual requirements. Advantageously, the dry powdercomposition according to the invention provides excellent storagestability.

In particular, the invention provides a dry powder compositioncomprising a long-chain RNA molecule, wherein the long-chain RNAmolecule preferably comprises at least 30 nucleotides. Preferably, thelong-chain RNA molecule is a molecule as defined herein. Morepreferably, the long-chain RNA molecule is not an RNA molecule selectedfrom the group consisting of a small interfering RNA (siRNA), amicroRNA, a small nuclear RNA (snRNA), a small-hairpin (sh) RNA, ariboswitch, a ribozyme or an aptamer. Even more preferably, thelong-chain RNA is not an siRNA, most preferably not a double-strandedsiRNA.

In a preferred embodiment, the dry powder composition as describedherein does not comprise an RNA molecule comprising less than 30nucleotides, less than 200 nucleotides or less than 250 nucleotides. Thedry powder composition does preferably not comprise an RNA moleculeselected from the group consisting of a small interfering RNA (siRNA),preferably a single-stranded or a double-stranded siRNA, a microRNA, asmall nuclear RNA (snRNA), a small-hairpin (sh) RNA, a riboswitch, aribozyme or an aptamer. Even more preferably, the dry powder compositiondoes not comprise an siRNA, most preferably not a double-stranded siRNA.

With respect to the following description of the inventive dry powdercomposition it is noted that the definitions and specifications providedtherein may also be applied to the inventive method, which issubsequently described as another aspect of the invention.

In the context of the present invention, the term “dry powder” (or “drypowder composition”) typically refers to a composition that is—amongstother features—characterized by its residual moisture content, which ispreferably low enough in order to prevent the formation of aggregatesthat would reduce or inhibit the flowability of the powder. As usedherein, the term “residual moisture content” (or “residual moisture”)typically refers to the total amount of solvent present in the drypowder composition. Said total amount of residual solvents in the drypowder composition is determined using any suitable method known in theart. For example, methods for determining the residual moisture contentcomprise the Karl-Fischer-titrimetric technique or the thermalgravimetric analysis (TGA) method. In a preferred embodiment, theresidual solvent comprised in the dry powder composition is water or anessentially aqueous solution and the residual moisture contentcorresponds to the residual water content of the dry powder composition,which is determined by the Karl-Fischer-titrimetric technique. Withoutbeing bound by any theory, the low residual moisture content of theinventive dry powder composition is expected to contribute to itsexcellent storage stability.

Preferably, the residual moisture content of the dry powder compositionaccording to the invention is 15% (w/w) or less, more preferably 10%(w/w) or less, even more preferably 9% (w/w), 8% (w/w), 7% (w/w), 6%(w/w) or 5% (w/w). In a preferred embodiment, the residual moisturecontent of the dry powder composition is 5% (w/w) or less, preferably 4%(w/w) or less. In a particularly preferred embodiment, the residualmoisture content is 7% (w/w) or less. In a further preferred embodiment,the residual moisture content of the dry powder composition in the rangefrom 0% to 15% (w/w), from 0% to 10% (w/w), from 0% to 7% (w/w), from 0%to 5% (w/w), from 0% to 4% (w/w), from 3% to 6% (w/w) or from 2% to 5%(w/w).

In a further preferred embodiment, the residual moisture content of thedry powder composition as described herein is 5% (w/w) or less, morepreferably 4% (w/w) or less, even more preferably 3% (w/w) or less, 2%(w/w) or less, or 1% (w/w) or less. Alternatively, the residual moisturecontent of the dry powder composition as described herein is preferablyin the range from 0% to 5% (w/w), from 0% to 4% (w/w), from 0% to 3%(w/w), from 0% to 2% (w/w) or from 0% to 1% (w/w).

In a further preferred embodiment, the present invention provides a drypowder composition having a residual water content of 15% (w/w) or less,more preferably 10% (w/w) or less, even more preferably 9% (w/w), 8%(w/w), 7% (w/w), 6% (w/w) or 5% (w/w). According to a preferredembodiment, the residual water content of the dry powder composition is5% (w/w) or less, preferably 4% (w/w) or less. In a particularlypreferred embodiment, the residual water is 7% (w/w) or less. In afurther preferred embodiment, the residual water content of the drypowder composition in the range from 0% to 15% (w/w), from 0% to 10%(w/w), from 0% to 7% (w/w), from 0% to 5% (w/w), from 0% to 4% (w/w),from 3% to 6% (w/w) or from 2% to 5% (w/w).

Preferably, the dry powder composition comprising a long-chain RNAmolecule comprises a plurality of particles. Therein, the term“particle” typically refers to an individual solid particle of the drypowder composition. The individual particles of the dry powdercomposition according to the invention are preferably physicallyseparated from each other, i.e. the individual particles that constitutethe dry powder may be in lose and reversible contact with each other (asopposed to an irreversible link between individual particles).Preferably, the term “particle” refers to the smallest physical entityof the inventive dry powder composition. The particles of the inventivedry powder composition do preferably not stick to each other. Theparticulate nature of the formulation contributes to the superiorcharacteristics of the inventive dry powder composition, e.g. its freeflowability.

In a preferred embodiment, the plurality of individual particles of thedry powder composition according to the invention is characterized by asize distribution, wherein the size of individual particles may be thesame or different from each other. Typically, the size of the particlesof a dry powder composition is characterized by a Gaussian distributionor a quasi-Gaussian distribution. Preferably, the dry powder compositionaccording to the invention is characterized by a volume weightedparticle size distribution as determined, for instance, by static lightscattering techniques, such as laser diffraction, or by using a cascadeimpactor. In a volume weighted distribution, the contribution of eachparticle in the dry powder composition relates to the volume of thatparticle. The dry powder composition according to the invention ispreferably characterized by parameters such as, for example, Dv10, Dv50,Dv90 or the mass median aerodynamic diameter (MMAD).

The parameter “Dv50” (or “volume D50” or “volume weighted D50”) relatesto the median particle size based on a volume weighted particle sizedistribution. Dv50 thus typically describes the particle size (based ona volume weighted distribution), preferably the diameter of a particlein micrometers (μm), with 50% of the particles in the distributionhaving a larger size and 50% of the particles in the distribution havinga smaller size than Dv50. In a volume weighted distribution, theparameter “Dv50” typically relates to the diameter (e.g. in micrometers)of a hypothetical spherical particle, which has the volume of thecorresponding actual particle in the distribution (which may or may notbe spherical).

Analogously, the parameter “Dv10” corresponds to the cut-off size(preferably in μm) of the particles in a volume weighted distribution,which represent 10% of the total volume of the sample, and which have aparticle size equal to or smaller than the Dv10 value. Preferably, theDv10 of the dry powder composition according to the invention is atleast 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm. Morepreferably, the Dv10 of the dry powder composition is equal to or lowerthan 5, equal to or lower than 10, or equal to or lower than 20 μm. Mostpreferably, the Dv10 is in a range from 1 μm to 10 μm or from about 3 μmto about 5 μm.

The parameter “Dv90” corresponds to the cut-off size (preferably in μm)of the particles in a volume weighted distribution, which represent 90%of the total volume of the sample, and which have a particle size equalto or smaller than the Dv90 value. Preferably, the Dv90 of the drypowder composition according to the invention is at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 μm. Preferably, theDv90 of the dry powder composition according to the invention is equalto or lower than 1.500, 1.250, 1.000, 750, 600, 500, 400, 300, 200 or100 μm. More preferably, the Dv90 may be in a range from 0.3 μm to 2.000μm, from 1 μm to 1.000 μm, from 2 μm to 500 μm or from 2 μm to 200 μm.In a preferred embodiment, the Dv90 of the dry powder composition is atleast 1 μm or in the range from 1 to 200 μm. In a particularly preferredembodiment, the Dv90 of the dry powder composition is at least 3 μm, atleast 5 μm or at least 20 μm.

The mass median aerodynamic diameter (MMAD) describes the particle sizebased on the aerodynamic properties of the respective particle, inparticular its settling behaviour. The MMAD is preferably determinedusing any suitable instrument known in the art, such as, for instance,an APS™ spectrometer (TSI Inc.). The MMAD relates to the medianaerodynamic diameter of the particle distribution and is the diameter ofa unit density sphere having the same settling velocity, in air, as theparticle. The MMAD thus typically describes the particle size,preferably in micrometers (μm), with 50% of the particles in thedistribution having a larger size and 50% of the particles in thedistribution having a smaller size than the MMAD value. Preferably, thedry powder composition according to the invention is characterized by aMMAD of at least 0.3 μm. Alternatively, the MMAD of the dry powdercomposition according to the invention is at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 μm. Preferably, the MMAD ofthe dry powder composition according to the invention is equal to orless than 1.500, 1.250, 1.000, 750, 600, 500, 400, 300, 200 or 100 μm.Further preferably, the MMAD may be in a range from 0.3 μm to 2.000 μm,from 1 μm to 1.000 μm, from 2 μm to 500 μm or from 2 μm to 200 μm. In apreferred embodiment, the MMAD of the dry powder composition is at least1 μm or in the range from 1 to 200 μm. In a particularly preferredembodiment, the MMAD of the dry powder composition is at least 3 μm, atleast 5 μm or at least 20 μm.

The dry powder composition is preferably characterized by using the spanof the particle size distribution as a parameter. Therein, the span (fora volume weighted distribution) is defined according to the followingformula

${Span} = \frac{{{Dv}\; 90} - {{Dv}\; 10}}{{Dv}\; 50}$

For distributions, which are not volume weighted, the Dv values in theformula above are respectively replaced by the corresponding Dx values,e.g. Dn90 for a number weighted distribution. In a preferred embodiment,the inventive dry powder composition is characterized by a low spanvalue, which indicates a narrow (or more homogeneous) particle sizedistribution. Typically, a narrow distribution enhances the flowabilityof the dry powder composition. Preferably, the span of the dry powdercomposition according to the present invention is equal to or less than5, more preferably equal to or less than 4, and even more preferablyequal to or less than 3. In a particularly preferred embodiment, theparticle size distribution of the dry powder composition according tothe invention is characterized by a span of equal to or less than about2 or a span of equal to or less than about 1.5.

In a preferred embodiment, the dry powder composition comprises aplurality of spherical particles. As used herein, the term “spherical”comprises not only geometrically perfect spheres, but also moreirregular shapes, such as spheroidal, elipsoid, oval or roundedparticles. The shape of an individual particle can be determined byknown methods and by using instruments, which are commerciallyavailable, such as Lasentec™ (particle chord length FBRM), Malvern™(Fraunhofer diffraction) or Coulter Counter™ (electric zone sensing).Typically, the volume and the surface area of an individual particle aredetermined. By using such parameters, Waddell's sphericity ψ (hereinalso referred to as “sphericity” or “circularity”) may be calculated,e.g. by using the following formula

$\psi = \frac{{surfa}\; {ce}\mspace{14mu} {area}\mspace{14mu} {of}\mspace{14mu} {sphere}\mspace{14mu} {of}\mspace{14mu} {equal}\mspace{14mu} {volume}\mspace{14mu} {to}\mspace{14mu} {the}{\mspace{11mu} \;}{particle}}{{surface}\mspace{14mu} {area}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {particle}}$

It is preferred that the average sphericity of the particles, which arecontained in the inventive dry powder composition, is at least 0.7,preferably at least 0.75, at least 0.8, at least 0.85, at least 0.9, atleast 0.95 or 1. Preferably, the average sphericity of the particles,which are contained in the dry powder composition, is in the range from0.7 to 1, more preferably in the range from 0.8 to 1, from 0.85 to 1, orfrom 0.9 to 1. In a particularly preferred embodiment, the averagesphericity of the particles, which are contained in the inventive drypowder composition, is in the range from 0.7 to 1.

In another preferred embodiment, the dry powder composition according tothe invention consists of particles, which are characterized by asphericity of at least 0.7, preferably at least 0.75, at least 0.8, atleast 0.85, at least 0.9, at least 0.95 or 1. Preferably, the dry powdercomposition consists of particles with a sphericity in the range from0.7 to 1, more preferably in the range from 0.8 to 1, from 0.85 to 1, orfrom 0.9 to 1.

Alternatively, the sphericity of those particles of the dry powdercomposition that have a particle size equal to Dv50 as defined herein isat least 0.7, preferably at least 0.75, at least 0.8, at least 0.85, atleast 0.9, at least 0.95 or 1. Preferably, the sphericity of thoseparticles of the dry powder composition that have a particle size equalto Dv50 as defined herein is in the range from 0.7 to 1, more preferablyin the range from 0.8 to 1, from 0.85 to 1, or from 0.9 to 1. Even morepreferably, those particles of the dry powder composition that have aparticle size equal to Dv90 as defined herein have a sphericity of atleast 0.7, preferably of at least 0.75, at least 0.8, at least 0.85, atleast 0.9, at least 0.95 or 1. Preferably, the sphericity of thoseparticles of the dry powder composition that have a particle size equalto Dv90 as defined herein is in the range from 0.7 to 1, more preferablyin the range from 0.8 to 1, from 0.85 to 1, or from 0.9 to 1.

Further preferably, the average sphericity of those particles of the drypowder composition that have a particle size equal to or lower than Dv50as defined herein is at least 0.7, preferably at least 0.75, at least0.8, at least 0.85, at least 0.9, at least 0.95 or 1. Preferably, theaverage sphericity of those particles of the dry powder composition thathave a particle size equal to or lower than Dv50 as defined herein is inthe range from 0.7 to 1, more preferably in the range from 0.8 to 1,from 0.85 to 1, or from 0.9 to 1. Even more preferably, the averagesphericity of those particles of the dry powder composition that have aparticle size equal to or lower than Dv90 as defined herein is at least0.7, preferably of at least 0.75, at least 0.8, at least 0.85, at least0.9, at least 0.95 or 1. Preferably, the average sphericity of thoseparticles of the dry powder composition that have a particle size equalto or lower than Dv90 as defined herein is in the range from 0.7 to 1,more preferably in the range from 0.8 to 1, from 0.85 to 1, or from 0.9to 1.

In the context of the present invention, the term “RNA” is used asabbreviation for ribonucleic acid. RNA is a nucleic acid molecule, i.e.a polymer consisting of nucleotides. These nucleotides are usuallyadenosine-monophosphate, uridine-monophosphate, guanosine-monophosphateand cytidine-monophosphate monomers, which are connected to each otheralong a so-called backbone. The backbone is formed by phosphodiesterbonds between the sugar, i.e. ribose, of a first and a phosphate moietyof a second, adjacent monomer. The specific succession of the monomersis called the RNA sequence. As used herein, the term “RNA molecule” isnot limited to any particular type of RNA.

For example, RNA may be obtainable by transcription of a DNA-sequence,e.g., inside a cell. In eukaryotic cells, transcription is typicallyperformed inside the nucleus or the mitochondria. In vivo, transcriptionof DNA usually results in the so-called premature RNA, which has to beprocessed into so-called messenger-RNA, usually abbreviated as mRNA.Processing of the premature RNA, e.g. in eukaryotic organisms, comprisesa variety of different posttranscriptional-modifications such assplicing, 5′-capping, polyadenylation, export from the nucleus or themitochondria and the like. The sum of these processes is also calledmaturation of RNA. The mature messenger RNA usually provides thenucleotide sequence that may be translated into an amino acid sequenceof a particular peptide or protein. Typically, a (mature) mRNA comprisesa 5′-cap, optionally a 5′UTR, an open reading frame, optionally a 3′UTRand a poly(A) and/or poly(C) sequence. Furthermore, the term “RNAmolecule” comprises ribonucleic acids comprising more than one openreading frame, such as bicistronic or multicistronic RNA molecules. Abicistronic or multicistronic RNA molecule is typically an RNA molecule,preferably an mRNA molecule, that may typically have two (bicistronic)or more (multicistronic) open reading frames (ORF).

Aside from messenger RNA, several non-coding types of RNA exist, whichmay be involved in regulation of transcription and/or translation, suchas a ribosomal RNA (rRNA) or a transfer RNA (tRNA). The terms “RNA” or“RNA molecule” further encompass other coding RNA molecules, such asviral RNA, retroviral RNA, self-replicating RNA (replicon RNA), smallinterfering RNA (siRNA), microRNA, small nuclear RNA (snRNA),small-hairpin (sh) RNA, riboswitches, ribozymes or an aptamers.

The term “long-chain RNA molecule” (or “long-chain RNA”) as used hereintypically refers to an RNA molecule, preferably as described herein,which preferably comprises at least 30 nucleotides. Alternatively, thelong-chain RNA molecule according to the invention may comprise at least35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250,300, 350, 400, 450 or at least 500 nucleotides. In a preferredembodiment, the long-chain RNA molecule comprises at least 100nucleotides, even more preferably at least 200 nucleotides. Thelong-chain RNA molecule further preferably comprises from 30 to 50.000nucleotides, from 30 to 20.000 nucleotides, from 100 to 20.000nucleotides, from 200 to 20.000 nucleotides, from 250 to 15.000nucleotides or from 500 to 20.000 nucleotides.

According to a preferred embodiment, the long-chain RNA of the drypowder composition as described herein comprises more than 200nucleotides, preferably at least 250 nucleotides. Alternatively, thelong-chain RNA as described herein may comprise more than 210, more than220, more than 230, more than 240, more than 250, more than 260, morethan 270, more than 280, more than 290, more than 300, more than 350,more than 400, more than 450 or more than 500 nucleotides. Morepreferably, the long-chain RNA as described herein may comprise at leastabout 210, at least about 220, at least about 230, at least about 240,at least about 250, at least about 260, at least about 270, at leastabout 280, at least about 290, at least about 300, at least about 350,at least about 400, at least about 450 or at least about 500nucleotides.

The inventive dry powder composition comprises a (first) long-chain RNAmolecule and may further comprise a second or further RNA molecule,which may also be a long-chain RNA molecule, preferably as definedherein. Preferably, the second or further RNA molecule comprised in thedry powder composition is distinct from the (first) long-chain RNAmolecule.

In a preferred embodiment, the long-chain RNA molecule comprised in thedry powder composition according to the invention is not an RNA moleculeselected from the group consisting of a small interfering RNA (siRNA), amicroRNA, a small nuclear RNA (snRNA), a small-hairpin (sh) RNA orriboswitch, a ribozyme, and an aptamer. More preferably, the long-chainRNA as described herein is not an siRNA, most preferably not adouble-stranded siRNA.

As used herein, the term “RNA molecule” typically refers to asingle-stranded or a double-stranded RNA molecule. In a preferredembodiment, the long-chain RNA molecule of the inventive dry powdercomposition is a single-stranded RNA molecule.

In a further embodiment, the long-chain RNA molecule comprised in thedry powder composition according to the invention is a coding RNAmolecule or an immunostimulatory RNA molecule.

In a preferred embodiment, the long-chain RNA is a coding RNA, whichcomprises at least one open reading frame encoding a peptide or protein.

In the context of the present invention, the long-chain RNA molecule maybe a coding RNA molecule encoding a protein or a peptide, which may beselected, without being restricted thereto, e.g. from therapeuticallyactive proteins or peptides, selected e.g. from adjuvant proteins, fromantigens, e.g. tumour antigens, pathogenic antigens (e.g. selected, fromanimal antigens, from viral antigens, from protozoan antigens, frombacterial antigens), allergenic antigens, autoimmune antigens, orfurther antigens, preferably as defined herein, from allergens, fromantibodies, from immunostimulatory proteins or peptides, fromantigen-specific T-cell receptors, or from any other protein or peptidesuitable for a specific (therapeutic) application, wherein the codingRNA molecule may be transported into a cell, a tissue or an organism andthe protein may be expressed subsequently in this cell, tissue ororganism. In a particularly preferred embodiment, the long-chain RNAmolecule is an mRNA molecule.

The long-chain RNA molecule of the dry powder composition may further bean immunostimulatory RNA molecule, such as any RNA molecule known in theart, which is capable of inducing an innate immune response.Particularly preferred in this context are immunostimulatory RNAmolecules as described in WO 2009/095226.

The dry powder composition may further comprise a modified RNA molecule.In a preferred embodiment, the long-chain RNA molecule comprises atleast one modification as described herein. Alternatively oradditionally, the dry powder composition may comprise a second orfurther RNA molecule (distinct from the (first) long-chain RNAmolecule), which comprises at least one modification as describedherein. Preferably, the long-chain RNA molecule of the dry powdercomposition according to the invention comprises an RNA modification,which preferably increases the stability of the RNA molecule and/or theexpression of a protein encoded by the RNA molecule. Several RNAmodifications are known in the art, which can be applied to an RNAmolecule in the context of the present invention.

Chemical Modifications:

The term “RNA modification” as used herein may refer to chemicalmodifications comprising backbone modifications as well as sugarmodifications or base modifications.

In this context, a modified RNA molecule as defined herein may containnucleotide analogues/modifications, e.g. backbone modifications, sugarmodifications or base modifications. A backbone modification inconnection with the present invention is a modification, in whichphosphates of the backbone of the nucleotides contained in an RNAmolecule as defined herein are chemically modified. A sugar modificationin connection with the present invention is a chemical modification ofthe sugar of the nucleotides of the RNA molecule as defined herein.Furthermore, a base modification in connection with the presentinvention is a chemical modification of the base moiety of thenucleotides of the RNA molecule. In this context, nucleotide analoguesor modifications are preferably selected from nucleotide analogues,which are applicable for transcription and/or translation.

Sugar Modifications:

The modified nucleosides and nucleotides, which may be incorporated intoa modified RNA molecule as described herein, can be modified in thesugar moiety. For example, the 2′ hydroxyl group (OH) can be modified orreplaced with a number of different “oxy” or “deoxy” substituents.Examples of “oxy”-2′ hydroxyl group modifications include, but are notlimited to, alkoxy or aryloxy (—OR, e.g., R═H, alkyl, cycloalkyl, aryl,aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG),—O(CH₂CH₂O)nCH₂CH₂OR; “locked” nucleic acids (LNA) in which the 2′hydroxyl is connected, e.g., by a methylene bridge, to the 4′ carbon ofthe same ribose sugar; and amino groups (—O-amino, wherein the aminogroup, e.g., NRR, can be alkylamino, dialkylamino, heterocyclyl,arylamino, diarylamino, heteroarylamino, or diheteroaryl amino, ethylenediamine, polyamino) or aminoalkoxy.

“Deoxy” modifications include hydrogen, amino (e.g. NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,diheteroaryl amino, or amino acid); or the amino group can be attachedto the sugar through a linker, wherein the linker comprises one or moreof the atoms C, N, and O.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, a modified RNA molecule can include nucleotidescontaining, for instance, arabinose as the sugar.

Backbone Modifications:

The phosphate backbone may further be modified in the modifiednucleosides and nucleotides, which may be incorporated into a modifiedRNA molecule as described herein. The phosphate groups of the backbonecan be modified by replacing one or more of the oxygen atoms with adifferent substituent. Further, the modified nucleosides and nucleotidescan include the full replacement of an unmodified phosphate moiety witha modified phosphate as described herein. Examples of modified phosphategroups include, but are not limited to, phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. Phosphorodithioates have both non-linking oxygensreplaced by sulfur. The phosphate linker can also be modified by thereplacement of a linking oxygen with nitrogen (bridgedphosphoroamidates), sulfur (bridged phosphorothioates) and carbon(bridged methylene-phosphonates).

Base Modifications:

The modified nucleosides and nucleotides, which may be incorporated intoa modified RNA molecule as described herein can further be modified inthe nucleobase moiety. Examples of nucleobases found in RNA include, butare not limited to, adenine, guanine, cytosine and uracil. For example,the nucleosides and nucleotides described herein can be chemicallymodified on the major groove face. In some embodiments, the major groovechemical modifications can include an amino group, a thiol group, analkyl group, or a halo group.

In particularly preferred embodiments of the present invention, thenucleotide analogues/modifications are selected from base modifications,which are preferably selected from2-amino-6-chloropurineriboside-5′-triphosphate,2-Aminopurine-riboside-5′-triphosphate;2-aminoadenosine-5′-triphosphate,2′-Amino-2′-deoxycytidine-triphosphate, 2-thiocytidine-5′-triphosphate,2-thiouridine-5′-triphosphate, 2′-Fluorothymidine-5′-triphosphate,2′-O-Methyl inosine-5′-triphosphate 4-thiouridine-5′-triphosphate,5-aminoallylcytidine-5′-triphosphate,5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate,5-bromouridine-5′-triphosphate,5-Bromo-2′-deoxycytidine-5′-triphosphate,5-Bromo-2′-deoxyuridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate,5-Iodo-2′-deoxycytidine-5′-triphosphate, 5-iodouridine-5′-triphosphate,5-Iodo-2′-deoxyuridine-5′-triphosphate,5-methylcytidine-5′-triphosphate, 5-methyluridine-5′-triphosphate,5-Propynyl-2′-deoxycytidine-5′-triphosphate,5-Propynyl-2′-deoxyuridine-5′-triphosphate,6-azacytidine-5′-triphosphate, 6-azauridine-5′-triphosphate,6-chloropurineriboside-5′-triphosphate,7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate,benzimidazole-riboside-5′-triphosphate,N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate,N6-methyladenosine-5′-triphosphate, O6-methylguanosine-5′-triphosphate,pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate,xanthosine-5′-triphosphate. Particular preference is given tonucleotides for base modifications selected from the group ofbase-modified nucleotides consisting of5-methylcytidine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,5-bromocytidine-5′-triphosphate, and pseudouridine-5′-triphosphate.

In some embodiments, modified nucleosides include pyridin-4-oneribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine,4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine,3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

In some embodiments, modified nucleosides include 5-aza-cytidine,pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

In other embodiments, modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine,7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine,7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine,1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine,N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

In other embodiments, modified nucleosides include inosine,1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

In some embodiments, the nucleotide can be modified on the major grooveface and can include replacing hydrogen on C-5 of uracil with a methylgroup or a halo group. In specific embodiments, a modified nucleoside is5′-O-(1-Thiophosphate)-Adenosine, 5′-O-(1-Thiophosphate)-Cytidine,5′-O-(1-Thiophosphate)-Guanosine, 5′-O-(1-Thiophosphate)-Uridine or5′-O-(1-Thiophosphate)-Pseudouridine.

In further specific embodiments, a modified RNA may comprise nucleosidemodifications selected from 6-aza-cytidine, 2-thio-cytidine,α-thio-cytidine, Pseudo-iso-cytidine, 5-aminoallyl-uridine,5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine,α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine,deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine,α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytdine, 8-oxo-guanosine,7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-Chloro-purine,N6-methyl-2-amino-purine, Pseudo-iso-cytidine, 6-Chloro-purine,N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine,7-deaza-adenosine.

Lipid Modification:

According to a further embodiment, a modified RNA molecule as definedherein can contain a lipid modification. Such a lipid-modified RNAmolecule typically comprises an RNA as defined herein. Such alipid-modified RNA molecule as defined herein typically furthercomprises at least one linker covalently linked with that RNA molecule,and at least one lipid covalently linked with the respective linker.Alternatively, the lipid-modified RNA molecule comprises at least oneRNA molecule as defined herein and at least one (bifunctional) lipidcovalently linked (without a linker) with that RNA molecule. Accordingto a third alternative, the lipid-modified RNA molecule comprises an RNAmolecule as defined herein, at least one linker covalently linked withthat RNA molecule, and at least one lipid covalently linked with therespective linker, and also at least one (bifunctional) lipid covalentlylinked (without a linker) with that RNA molecule. In this context, it isparticularly preferred that the lipid modification is present at theterminal ends of a linear RNA sequence.

Modification of the 5′-End of a Modified RNA Molecule:

According to another preferred embodiment of the invention, a modifiedRNA molecule as defined herein, can be modified by the addition of aso-called “5′ CAP” structure.

A 5′-cap is an entity, typically a modified nucleotide entity, whichgenerally “caps” the 5′-end of a mature mRNA. A 5′-cap may typically beformed by a modified nucleotide, particularly by a derivative of aguanine nucleotide. Preferably, the 5′-cap is linked to the 5′-terminusvia a 5′-5′-triphosphate linkage. A 5′-cap may be methylated, e.g.m7GpppN, wherein N is the terminal 5′ nucleotide of the nucleic acidcarrying the 5′-cap, typically the 5′-end of an RNA. m7GpppN is the5′-CAP structure which naturally occurs in mRNA transcribed bypolymerase II and is therefore not considered as modification comprisedin a modified RNA in this context. Accordingly, a modified RNA of thepresent invention may comprise a m7GpppN as 5′-CAP, but additionally themodified RNA comprises at least one further modification as definedherein.

Further examples of 5′cap structures include glyceryl, inverted deoxyabasic residue (moiety), 4′,5′ methylene nucleotide,1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclicnucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides,alpha-nucleotide, modified base nucleotide, threo-pentofuranosylnucleotide, acyclic 3′,4′-seco nucleotide, acyclic 3,4-dihydroxybutylnucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3′-3′-invertednucleotide moiety, 3′-3′-inverted abasic moiety, 3′-2′-invertednucleotide moiety, 3′-2′-inverted abasic moiety, 1,4-butanediolphosphate, 3′-phosphoramidate, hexylphosphate, aminohexyl phosphate,3′-phosphate, 3′phosphorothioate, phosphorodithioate, or bridging ornon-bridging methylphosphonate moiety. These modified 5′-CAP structuresare regarded as at least one modification in this context.

Particularly preferred modified 5′-CAP structures are CAP1 (methylationof the ribose of the adjacent nucleotide of m7G), CAP2 (methylation ofthe ribose of the 2nd nucleotide downstream of the m7G), CAP3(methylation of the ribose of the 3rd nucleotide downstream of the m7G),CAP4 (methylation of the ribose of the 4th nucleotide downstream of them7G), ARCA (anti-reverse CAP analogue, modified ARCA (e.g.phosphothioate modified ARCA), inosine, N1-methyl-guanosine,2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

In a preferred embodiment, the inventive dry powder compositioncomprises a modified RNA molecule having at least one open readingframe, which encodes at least one peptide or protein. Said modified RNAmolecule having at least one open reading frame may be the (first)long-chain RNA molecule, preferably a long-chain mRNA molecule, or asecond or further RNA molecule, which may be comprised in the dry powdercomposition in addition to the (first) long-chain RNA molecule.Preferably, the sequence of the open reading frame in such an RNAmolecule is modified as described herein.

Modification of the G/C Content:

In a particularly preferred embodiment of the present invention, the G/Ccontent of the coding region of a modified RNA comprised in theinventive dry powder composition, is modified, particularly increased,compared to the G/C content of its particular wild type coding region,i.e. the unmodified coding region. The encoded amino acid sequence ofthe coding region is preferably not modified compared to the coded aminoacid sequence of the particular wild type coding region. Themodification of the G/C-content of the coding region of the modified RNAas defined herein is based on the fact that the sequence of any mRNAregion to be translated is important for efficient translation of thatmRNA. Thus, the composition and the sequence of various nucleotides areimportant. In particular, mRNA sequences having an increased G(guanosine)/C (cytosine) content are more stable than mRNA sequenceshaving an increased A (adenosine)/U (uracil) content. According to theinvention, the codons of the coding region are therefore varied comparedto its wild type coding region, while retaining the translated aminoacid sequence, such that they include an increased amount of G/Cnucleotides. In respect to the fact that several codons code for one andthe same amino acid (so-called degeneration of the genetic code), themost favourable codons for the stability can be determined (so-calledalternative codon usage). Depending on the amino acid to be encoded bythe coding region of the modified RNA as defined herein, there arevarious possibilities for modification of the RNA sequence, e.g. thecoding region, compared to its wild type coding region. In the case ofamino acids, which are encoded by codons, which contain exclusively G orC nucleotides, no modification of the codon is necessary. Thus, thecodons for Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG) and Gly(GGC or GGG) require no modification, since no A or U is present. Incontrast, codons which contain A and/or U nucleotides can be modified bysubstitution of other codons which code for the same amino acids butcontain no A and/or U. Examples of these are: the codons for Pro can bemodified from CCU or CCA to CCC or CCG; the codons for Arg can bemodified from CGU or CGA or AGA or AGG to CGC or CGG; the codons for Alacan be modified from GCU or GCA to GCC or GCG; the codons for Gly can bemodified from GGU or GGA to GGC or GGG. In other cases, although A or Unucleotides cannot be eliminated from the codons, it is however possibleto decrease the A and U content by using codons, which contain a lowercontent of A and/or U nucleotides. Examples of these are: the codons forPhe can be modified from UUU to UUC; the codons for Leu can be modifiedfrom UUA, UUG, CUU or CUA to CUC or CUG; the codons for Ser can bemodified from UCU or UCA or AGU to UCC, UCG or AGC; the codon for Tyrcan be modified from UAU to UAC; the codon for Cys can be modified fromUGU to UGC; the codon for His can be modified from CAU to CAC; the codonfor Gln can be modified from CAA to CAG; the codons for Ile can bemodified from AUU or AUA to AUC; the codons for Thr can be modified fromACU or ACA to ACC or ACG; the codon for Asn can be modified from AAU toAAC; the codon for Lys can be modified from AAA to AAG; the codons forVal can be modified from GUU or GUA to GUC or GUG; the codon for Asp canbe modified from GAU to GAC; the codon for Glu can be modified from GAAto GAG; the stop codon UAA can be modified to UAG or UGA. In the case ofthe codons for Met (AUG) and Trp (UGG), on the other hand, there is nopossibility of sequence modification. The substitutions listed above canbe used either individually or in any possible combination to increasethe G/C content of the coding region of the modified RNA as definedherein, compared to its particular wild type coding region (i.e. theoriginal sequence). Thus, for example, all codons for Thr occurring inthe wild type sequence can be modified to ACC (or ACG).

Preferably, the G/C content of the coding region of the modified RNA asdefined herein is increased by at least 7%, more preferably by at least15%, particularly preferably by at least 20%, compared to the G/Ccontent of the wild type coding region. According to a specificembodiment at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, more preferably atleast 70%, even more preferably at least 80% and most preferably atleast 90%, 95% or even 100% of the substitutable codons in the codingregion encoding at least one peptide or protein, which comprises apathogenic antigen or a fragment, variant or derivative thereof, aresubstituted, thereby increasing the G/C content of said coding region.In this context, it is particularly preferable to increase the G/Ccontent of the coding region of the modified RNA as defined herein, tothe maximum (i.e. 100% of the substitutable codons), compared to thewild type coding region.

Codon Optimization:

According to the invention, a further preferred modification of thecoding region encoding at least one peptide or protein of a modified RNAas defined herein, is based on the finding that the translationefficiency is also determined by a different frequency in the occurrenceof tRNAs in cells. Thus, if so-called “rare codons” are present in thecoding region of the wild type RNA sequence, to an increased extent, themRNA is translated to a significantly poorer degree than in the casewhere codons coding for relatively “frequent” tRNAs are present. In thiscontext, the coding region of the modified RNA is preferably modifiedcompared to the corresponding wild type coding region such that at leastone codon of the wild type sequence, which codes for a tRNA which isrelatively rare in the cell, is exchanged for a codon, which codes for atRNA which is relatively frequent in the cell and carries the same aminoacid as the relatively rare tRNA. By this modification, the codingregion of the modified RNA as defined herein, is modified such thatcodons, for which frequently occurring tRNAs are available, areinserted. In other words, according to the invention, by thismodification all codons of the wild type coding region, which code for atRNA which is relatively rare in the cell, can in each case be exchangedfor a codon, which codes for a tRNA which is relatively frequent in thecell and which, in each case, carries the same amino acid as therelatively rare tRNA. Which tRNAs occur relatively frequently in thecell and which, in contrast, occur relatively rarely is known to aperson skilled in the art; cf. e.g. Akashi, Curr. Opin. Genet. Dev.2001, 11(6): 660-666. The codons which use for the particular amino acidthe tRNA which occurs the most frequently, e.g. the Gly codon, whichuses the tRNA which occurs the most frequently in the (human) cell, areparticularly preferred.

According to the invention, it is particularly preferable to link thesequential G/C content, which is increased, in particular maximized, inthe coding region of the modified RNA as defined herein, with the“frequent” codons without modifying the amino acid sequence of thepeptide or protein encoded by the coding region of the RNA sequence.This preferred embodiment allows provision of a particularly efficientlytranslated and stabilized (modified) RNA sequence as defined herein.

In the context of the present invention, the long-chain RNA molecule mayalso comprise a 5′- and/or 3′ untranslated region (5′-UTR or 3′-UTR,respectively). More preferably, the long-chain RNA molecule comprises a5′-CAP structure.

Preferably, the long-chain RNA molecule further comprises a poly(A)sequence. The length of the poly(A) sequence may vary. For example, thepoly(A) sequence may have a length of about 20 adenine nucleotides up toabout 300 adenine nucleotides, preferably of about 40 to about 200adenine nucleotides, more preferably from about 50 to about 100 adeninenucleotides, such as about 60, 70, 80, 90 or 100 adenine nucleotides.Most preferably, the long-chain RNA molecule comprises a poly(A)sequence of about 60 to about 70 nucleotides, most preferably 64 adeninenucleotides.

Preferably, the poly(A) sequence in the long-chain RNA molecule isderived from a DNA template by in vitro transcription. Alternatively,the poly(A) sequence may also be obtained in vitro by common methods ofchemical-synthesis without being necessarily transcribed from aDNA-progenitor.

Alternatively, the long-chain RNA molecule optionally comprises apolyadenylation signal, which is defined herein as a signal, whichconveys polyadenylation to a (transcribed) mRNA by specific proteinfactors (e.g. cleavage and polyadenylation specificity factor (CPSF),cleavage stimulation factor (CstF), cleavage factors I and II (CF I andCF II), poly(A) polymerase (PAP)). In this context, a consensuspolyadenylation signal is preferred comprising the NN(U/T)ANA consensussequence. In a particularly preferred aspect, the polyadenylation signalcomprises one of the following sequences: AA(U/T)AAA or A(U/T)(U/T)AAA(wherein uridine is usually present in RNA and thymidine is usuallypresent in DNA).

In addition or as an alternative to a poly(A) sequence as describedabove, the long-chain RNA molecule may also comprise a poly(C) sequence,preferably in the region 3′ of the coding region of the RNA. A poly(C)sequence is typically a stretch of multiple cytosine nucleotides,typically about 10 to about 200 cytidine nucleotides, preferably about10 to about 100 cytidine nucleotides, more preferably about 10 to about70 cytidine nucleotides or even more preferably about 20 to about 50 oreven about 20 to about 30 cytidine nucleotides. A poly(C) sequence maypreferably be located 3′ of the coding region comprised by a nucleicacid. In a preferred embodiment of the present invention, the long-chainRNA molecule comprises a poly(A) sequence and a poly(C) sequence,wherein the poly(C) sequence is located 3′ of the poly(A) sequence.

In a particularly preferred embodiment, the long-chain RNA molecule inthe context of the present invention comprises in 5′-to-3′-direction, a5′-UTR, an open reading frame, preferably a modified open reading frameas defined herein, a 3′-UTR element and a poly(A) or a poly(C) sequence.

According to a preferred embodiment, the inventive dry powdercomposition may comprise the long-chain RNA molecule as described hereinin free form (“naked RNA”) or in the form of a complex with anothercompound, such as a transfection or complexation agent. For example, thelong-chain RNA molecule may be present in the dry powder composition ina complex with a cationic or polycationic carrier or compound, which mayserve as transfection or complexation agent. In a preferred embodiment,the dry powder composition comprises both, the long-chain RNA in freeform as well in a complex with a cationic or polycationic carrier orcompound. Such a complex of long-chain RNA with a cationic orpolycationic carrier or compound may be present in the inventive drypowder composition or in an intermediate product as a nanoparticle,preferably as defined herein. The preparation of RNA complexes withpolycationic or cationic compounds is known in the art and is preferablycarried out as described in WO2010/037539 or WO2011/026641, the entiredisclosure of which is herewith incorporated by reference.

In this context, the long-chain RNA molecule in the inventive dry powdercomposition is preferably complexed by a compound selected from thegroup of polymers or complexing agents, typically comprising, withoutbeing limited thereto, any polymer suitable for the preparation of apharmaceutical composition, such as minor/major groove binders, nucleicacid binding proteins, lipoplexes, nanoplexes, non-cationic ornon-polycationic compounds, such as PLGA, Polyacetate, Polyacrylate,PVA, Dextran, hydroxymethylcellulose, starch, MMP, PVP, heparin, pectin,hyaluronic acid, and derivatives thereof, or cationic or polycationiccompound, particularly cationic or polycationic polymers or cationic orpolycationic lipids, preferably a cationic or polycationic polymers. Inthe context of the present invention, such a cationic or polycationiccompound is typically selected from any cationic or polycationiccompound, suitable for complexing and thereby stabilizing a long-chainRNA molecule as defined herein, e.g. by associating the RNA moleculewith the cationic or polycationic compound.

According to an alternative embodiment, the dry powder compositionaccording to the invention comprises the long-chain RNA as describedherein formulated together with one or more cationic or polycationiccompounds, preferably with cationic or polycationic polymers, cationicor polycationic peptides or proteins, e.g. protamine, cationic orpolycationic polysaccharides and/or cationic or polycationic lipids.

According to a preferred embodiment, the long-chain RNA as describedherein may be complexed with lipids to form one or more liposomes,lipoplexes, or lipid nanoparticles. Therefore, in one embodiment, thedry powder composition comprises liposomes, lipoplexes, and/or lipidnanoparticles comprising the long-chain RNA.

Lipid-based formulations have been increasingly recognized as one of themost promising delivery systems for RNA due to their biocompatibilityand their ease of large-scale production. Cationic lipids have beenwidely studied as synthetic materials for delivery of RNA. After mixingtogether, nucleic acids are condensed by cationic lipids to formlipid/nucleic acid complexes known as lipoplexes. These lipid complexesare able to protect genetic material from the action of nucleases and todeliver it into cells by interacting with the negatively charged cellmembrane. Lipoplexes can be prepared by directly mixing positivelycharged lipids at physiological pH with negatively charged nucleicacids.

Conventional liposomes consist of a lipid bilayer that can be composedof cationic, anionic, or neutral (phospho)lipids and cholesterol, whichencloses an aqueous core. Both the lipid bilayer and the aqueous spacecan incorporate hydrophobic or hydrophilic compounds, respectively.Liposome characteristics and behaviour in vivo can be modified byaddition of a hydrophilic polymer coating, e.g. polyethylene glycol(PEG), to the liposome surface to confer steric stabilization.Furthermore, liposomes can be used for specific targeting by attachingligands (e.g., antibodies, peptides, and carbohydrates) to its surfaceor to the terminal end of the attached PEG chains (Front Pharmacol. 2015Dec. 1; 6:286).

Liposomes are colloidal lipid-based and surfactant-based deliverysystems composed of a phospholipid bilayer surrounding an aqueouscompartment. They may present as spherical vesicles and can range insize from 20 nm to a few microns. Cationic lipid-based liposomes areable to complex with negatively charged nucleic acids via electrostaticinteractions, resulting in complexes that offer biocompatibility, lowtoxicity, and the possibility of the large-scale production required forin vivo clinical applications. Liposomes can fuse with the plasmamembrane for uptake; once inside the cell, the liposomes are processedvia the endocytic pathway and the genetic material is then released fromthe endosome/carrier into the cytoplasm. Liposomes have long beenperceived as drug delivery vehicles because of their superiorbiocompatibility, given that liposomes are basically analogs ofbiological membranes, and can be prepared from both natural andsynthetic phospholipids (Int J Nanomedicine. 2014; 9: 1833-1843).

Cationic liposomes have been traditionally the most commonly usednon-viral delivery systems for oligonucleotides, including plasmid DNA,antisense oligos, and siRNA/small hairpin RNA-shRNA). Cationic lipids,such as DOTAP, (1,2-dioleoyl-3-trimethylammonium-propane) and DOTMA(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methyl sulfate)can form complexes or lipoplexes with negatively charged nucleic acidsto form nanoparticles by electrostatic interaction, providing high invitro transfection efficiency. Furthermore, neutral lipid-basednanoliposomes for RNA delivery as e.g. neutral1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)-based nanoliposomeswere developed. (Adv Drug Deliv Rev. 2014 February; 66: 110-116.).

Therefore, in one embodiment the long-chain RNA of the dry powdercomposition as described herein is complexed with a cationic lipidand/or a neutral lipid and thereby forms liposomes, lipid nanoparticles,lipoplexes or neutral lipid-based nanoliposomes.

Particularly preferred complexation agents in this context are cationicor polycationic compounds, including protamine, nucleoline, spermine orspermidine, or other cationic peptides or proteins, such aspoly-L-lysine (PLL), poly-arginine, oligoarginines as defined above,such as Arg₇, Arg₈, Arg₉, Arg₇, H₃R₉, R₉H₃, H₃R₉H₃, YSSR₉SSY, (RKH)₄,Y(RKH)₂R, etc., basic polypeptides, cell penetrating peptides (CPPs),including HIV-binding peptides, HIV-1 Tat (HIV), Tat-derived peptides,Penetratin, VP22 derived or analog peptides, HSV VP22 (Herpes simplex),MAP, KALA or protein transduction domains (PTDs), PpT620, proline-richpeptides, arginine-rich peptides, lysine-rich peptides, MPG-peptide(s),Pep-1, L-oligomers, Calcitonin peptide(s), Antennapedia-derived peptides(particularly from Drosophila antennapedia), pAntp, plsl, FGF,Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(1), pVEC,hCT-derived peptides, SAP, or histones. In a particularly preferredembodiment, the dry powder composition according to the inventioncomprises protamin, wherein the long-chain RNA molecule is preferablycomplexed by protamine.

The dry powder composition according to the invention preferablycomprises a cationic or polycationic compound in solution and/or incomplex with the long-chain RNA molecule. More preferably, the inventivedry powder composition comprises a cationic or polycationic compound,preferably protamine, and the long-chain RNA molecule at a weight ratio(RNA:protamine, w/w) in a range from 1:10 to 10:1, more preferably from5:1 to 1:1, even more preferably from 3:1 to 1:1. Most preferably, theweight ratio of the long-chain RNA molecule to cationic or polycationiccompound, preferably protamine, in the composition is 2:1 (w/w).

Furthermore, such cationic or polycationic compounds or carriers may becationic or polycationic peptides or proteins, which preferably compriseor are additionally modified to comprise at least one —SH moiety.Preferably, a cationic or polycationic carrier is selected from cationicpeptides having the following sum formula (I):

{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)};  formula (I)

wherein l+m+n+o+x=3-100, and l, m, n or o independently of each other isany number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80,81-90 and 91-100 provided that the overall content of Arg (Arginine),Lys (Lysine), His (Histidine) and Orn (Ornithine) represents at least10% of all amino acids of the oligopeptide; and Xaa is any amino acidselected from native (=naturally occurring) or non-native amino acidsexcept of Arg, Lys, His or Orn; and x is any number selected from 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, provided, that theoverall content of Xaa does not exceed 90% of all amino acids of theoligopeptide. Any of amino acids Arg, Lys, His, Orn and Xaa may bepositioned at any place of the peptide. In this context, cationicpeptides or proteins in the range of 7-30 amino acids are particularpreferred.

Further, the cationic or polycationic peptide or protein, when definedaccording to formula {(Arg)_(l); (Lys)_(m); (His)_(n); (Orn)_(o);(Xaa)_(x)} (formula (I)) as shown above and which comprise or areadditionally modified to comprise at least one —SH moeity, may be,without being restricted thereto, selected from subformula (Ia):

{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa′)_(x)(Cys)_(y)}  subformula(Ia)

wherein (Arg)_(l); (Lys)_(m); (His)_(n); (Orn)_(o); and x are as definedherein, Xaa′ is any amino acid selected from native (=naturallyoccurring) or non-native amino acids except of Arg, Lys, His, Orn or Cysand y is any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70,71-80 and 81-90, provided that the overall content of Arg (Arginine),Lys (Lysine), His (Histidine) and Orn (Ornithine) represents at least10% of all amino acids of the oligopeptide. Further, the cationic orpolycationic peptide may be selected from subformula (Ib):

Cys₁{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)}Cys₂  subformula(Ib)

wherein empirical formula {(Arg)_(l); (Lys)_(m); (His)_(n); (Orn)_(o);(Xaa)_(x)} (formula (I)) is as defined herein and forms a core of anamino acid sequence according to (semiempirical) formula (IV) andwherein Cys₁ and Cys₂ are Cysteines proximal to, or terminal to(Arg)_(l); (Lys)_(m); (His)_(n); (Orn)_(o); (Xaa)_(x).

Further preferred cationic or polycationic compounds, which can be usedas transfection or complexation agent may include cationicpolysaccharides, for example chitosan, polybrene, cationic polymers,e.g. polyethyleneimine (PEI), cationic lipids, e.g. DOTMA:[1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE,di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE:Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS:Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethylhydroxyethyl ammonium bromide, DOTAP:dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:O,O-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride,CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammoniumchloride, CLIP6:rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]-trimethylammonium,CLIP9:rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium,oligofectamine, or cationic or polycationic polymers, e.g. modifiedpolyaminoacids, such as β-aminoacid-polymers or reversed polyamides,etc., modified polyethylenes, such as PVP(poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified acrylates,such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.,modified Amidoamines such as pAMAM (poly(amidoamine)), etc., modifiedpolybetaaminoester (PBAE), such as diamine end modified 1,4 butanedioldiacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such aspolypropylamine dendrimers or pAMAM based dendrimers, etc.,polyimine(s), such as PEI: poly(ethyleneimine), poly(propyleneimine),etc., polyallylamine, sugar backbone based polymers, such ascyclodextrin based polymers, dextran based polymers, chitosan, etc.,silan backbone based polymers, such as PMOXA-PDMS copolymers, etc.,blockpolymers consisting of a combination of one or more cationic blocks(e.g. selected from a cationic polymer as mentioned above) and of one ormore hydrophilic or hydrophobic blocks (e.g polyethyleneglycole); etc.

In this context, it is particularly preferred that the long-chain RNAmolecule of the inventive dry powder composition is complexed at leastpartially with a cationic or polycationic compound, preferably acationic protein or peptide. Partially means that only a part of thelong-chain RNA molecule is complexed with a cationic or polycationiccompound and that the rest of the long-chain RNA molecule is inuncomplexed form (“free”). Preferably the ratio of complexed long-chainRNA to free long-chain RNA is selected from a range of about 5:1 (w/w)to about 1:10 (w/w), more preferably from a range of about 4:1 (w/w) toabout 1:8 (w/w), even more preferably from a range of about 3:1 (w/w) toabout 1:5 (w/w) or 1:3 (w/w), and most preferably the ratio of complexedlong-chain RNA molecule to free long-chain RNA molecule is selected froma ratio of about 1:1 (w/w).

In the context of the present invention, a particle of the dry powdercomposition as defined herein, may thus comprise a long-chain RNAmolecule in free form or complexed by a cationic or polycationiccompound. In a preferred embodiment, a particle of the inventive drypowder composition as described herein comprises or consists oflong-chain RNA complexed by a cationic or polycationic compound, whereinthe complex is preferably present as a nanoparticle as defined herein.As used herein, the term “nanoparticle” typically refers to a complex ofthe long-chain RNA molecule with a complexation agent as defined herein,preferably with a cationic or polycationic compound.

In a preferred embodiment, upon reconstitution of the dry powder in asuitable solvent, the complexed long-chain RNA molecule as describedherein is present in the solvent in the form of nanoparticles. The sizeof the nanoparticle comprising or consisting of complexed long-chain RNAmolecule after reconstitution is preferably from 50 to 500 nm, morepreferably from 50 to 200 nm. In a particularly preferred embodiment,the particle size of the nanoparticle comprising or consisting ofcomplexed long-chain RNA molecule after reconstitution is from 75 to 180nm, more preferably from 100 to 150 nm.

Preferably, the nanoparticle comprising or consisting of complexedlong-chain RNA molecule is characterized by at least onephysico-chemical property. Suitable methods for determining aphysico-chemical property of the nanoparticle comprising or consistingof complexed long-chain RNA molecule are known in the art. Preferably, aphysico-chemical property of the nanoparticle comprising or consistingof complexed long-chain RNA molecule is determined by using a methodselected from the group consisting of measurement of turbidity, dynamiclight scattering (DLS), nanoparticle tracking analysis (NTA),determining the Zeta potential and micro-flow imaging (MFI). Morepreferably, such method is used to characterize the nanoparticlescomprised in a liquid composition obtained after reconstitution of theinventive dry powder in an appropriate solvent, preferably water, morepreferably water for injection.

In a preferred embodiment, a physico-chemical property of thenanoparticle comprising or consisting of complexed long-chain RNAmolecule is determined by nanoparticle tracking analysis (NTA). As usedherein, the term ‘nanoparticle tracking analysis’ or ‘NTA’ refers to amethod for analyzing particles in a liquid that relates the rate ofBrownian motion to particle size. Suitable NTA protocols are known inthe art and instruments for NTA are commercially available (such as theNanoSight instruments, e.g. NanoSight LM20, NanoSight, Amesbury, UK). Inpreferred embodiments, the mean size, preferably the mean size asdetermined by NTA, of the nanoparticles comprising or consisting ofcomplexed long-chain RNA molecule is equal to or larger than 100 nm,equal to or larger than 101 nm, equal to or larger than 102 nm, equal toor larger than 103 nm, equal to or larger than 104 nm, equal to orlarger than 105 nm, equal to or larger than 106 nm, equal to or largerthan 107 nm, equal to or larger than 108 nm, equal to or larger than 109nm, equal to or larger than 110 nm, equal to or larger than 111 nm,equal to or larger than 112 nm, equal to or larger than 113 nm, equal toor larger than 114 nm, equal to or larger than 115 nm, equal to orlarger than 116 nm, equal to or larger than 117 nm, equal to or largerthan 118 nm, equal to or larger than 119 nm, equal to or larger than 120nm, equal to or larger than 125 nm or equal to or larger than 130 nm. Ina particularly preferred embodiment, the mean size, preferably the meansize as determined by NTA, of the nanoparticles comprising or consistingof complexed long-chain RNA molecule is equal to or larger than 110 nm,more preferably equal to or larger than 120 nm, most preferably the meansize, preferably the mean size as determined by NTA, of thenanoparticles comprising or consisting of complexed long-chain RNAmolecule is equal to or larger than 130 nm. Alternatively, the meansize, preferably the mean size as determined by NTA, of thenanoparticles comprising or consisting of complexed long-chain RNAmolecule is in a range from 100 nm to 200 nm, preferably from 110 nm to150 nm.

According to a further preferred embodiment, the mode size, preferablythe mode size as determined by NTA, of the nanoparticles comprising orconsisting of complexed long-chain RNA molecule is equal to or largerthan 90 nm, equal to or larger than 91 nm, equal to or larger than 92nm, equal to or larger than 93 nm, equal to or larger than 94 nm, equalto or larger than 95 nm, equal to or larger than 96 nm, equal to orlarger than 97 nm, equal to or larger than 98 nm, equal to or largerthan 99 nm, equal to or larger than 100 nm, equal to or larger than 105nm, equal to or larger than 110 nm, equal to or larger than 115 nm,equal to or larger than 120 nm, equal to or larger than 125 nm or equalto or larger than 130 nm. According to a particularly preferredembodiment, the mode size, preferably the mode size as determined byNTA, of the nanoparticles comprising or consisting of complexedlong-chain RNA molecule is equal to or larger than 95 nm, morepreferably equal to or larger than 100 nm, most preferably equal to orlarger than 105 nm. Alternatively, the mode size, preferably the modesize as determined by NTA, of the nanoparticles comprising or consistingof complexed long-chain RNA molecule is preferably in a range from 95 nmto 150 nm, more preferably in a range from 100 nm to 140 nm.

In a further preferred embodiment, the D10 size, preferably the D10 sizeas determined by NTA, of the nanoparticles comprising or consisting ofcomplexed long-chain RNA molecule is equal to or larger than 70 nm,equal to or larger than 71 nm, equal to or larger than 72 nm, equal toor larger than 73 nm, equal to or larger than 74 nm, equal to or largerthan 75 nm, equal to or larger than 76 nm, equal to or larger than 77nm, equal to or larger than 78 nm, equal to or larger than 79 nm, equalto or larger than 80 nm, equal to or larger than 85 nm, equal to orlarger than 90 nm, equal to or larger than 95 nm, equal to or largerthan 100 nm, equal to or larger than 105 nm or equal to or larger than110 nm. It is particularly preferred that the D10 size, preferably theD10 size as determined by NTA, of the nanoparticles comprising orconsisting of complexed long-chain RNA molecule is equal to or largerthan 75 nm, more preferably equal to or larger than 80 nm, mostpreferably equal to or larger than 85 nm or 90 nm. Alternatively, theD10 size, preferably the D10 size as determined by NTA, of thenanoparticles comprising or consisting of complexed long-chain RNAmolecule is in a range from 70 nm to 140 nm, more preferably in a rangefrom 75 nm to 135 nm, most preferably in a range from 80 nm to 130 nm.

According to another embodiment, the D50 size, preferably the D50 sizeas determined by NTA, of the nanoparticles comprising or consisting ofcomplexed long-chain RNA molecule is equal to or larger than 100 nm,equal to or larger than 101 nm, equal to or larger than 102 nm, equal toor larger than 103 nm, equal to or larger than 104 nm, equal to orlarger than 105 nm, equal to or larger than 106 nm, equal to or largerthan 107 nm, equal to or larger than 108 nm, equal to or larger than 109nm, equal to or larger than 110 nm, equal to or larger than 115 nm,equal to or larger than 120 nm, equal to or larger than 125 nm, equal toor larger than 130 nm, equal to or larger than 130 nm or equal to orlarger than 135 nm. It is particularly preferred that the D50 size,preferably the D50 size as determined by NTA, of the nanoparticlescomprising or consisting of complexed long-chain RNA molecule is equalto or larger than 100 nm, more preferably equal to or larger than 105nm, most preferably equal to or larger than 110 nm, equal to or largerthan 115 nm, or equal to or larger than 120 nm. Alternatively, the D50size, preferably the D50 size as determined by NTA, of the nanoparticlescomprising or consisting of complexed long-chain RNA molecule is in arange from 100 nm to 150 nm, more preferably in a range from 105 nm to145 nm, most preferably in a range from 110 nm to 140 nm.

According to a preferred embodiment, the D90 size, preferably the D90size as determined by NTA, of the nanoparticles comprising or consistingof complexed long-chain RNA molecule is equal to or larger than 145 nm,equal to or larger than 146 nm, equal to or larger than 147 nm, equal toor larger than 148 nm, equal to or larger than 148 nm, equal to orlarger than 149 nm, equal to or larger than 150 nm, equal to or largerthan 151 nm, equal to or larger than 152 nm, equal to or larger than 153nm, equal to or larger than 154 nm, equal to or larger than 155 nm,equal to or larger than 160 nm, equal to or larger than 165 nm, equal toor larger than 170 nm, equal to or larger than 180 nm or equal to orlarger than 190 nm. It is particularly preferred that the D90 size,preferably the D90 size as determined by NTA, of the nanoparticlescomprising or consisting of complexed long-chain RNA molecule is equalto or larger than 150 nm, more preferably equal to or larger than 160nm, most preferably equal to or larger than 170 nm or 180 nm.Alternatively, the D90 size, preferably the D90 size as determined byNTA, of the nanoparticles comprising or consisting of complexedlong-chain RNA molecule is in a range from 140 nm to 210 nm, morepreferably in a range from 150 nm to 210 nm, most preferably in a rangefrom 160 nm to 200 nm.

In a preferred embodiment, a physico-chemical property of thenanoparticle comprising or consisting of complexed long-chain RNAmolecule is determined by dynamic light scattering (DLS). In thiscontext, the term ‘dynamic light scattering’ or ‘DLS’ refers to a methodfor analyzing particles in a liquid, wherein the liquid is typicallyilluminated with a monochromatic light source and wherein the lightscattered by particles in the liquid is detected. Due to Brownianmotion, smaller particles typically result in time-dependent scatteringintensity fluctuations that are distinct from those observed for largerparticles. DLS can thus be used to measure particle sizes in a liquid.Suitable DLS protocols are known in the art. DLS instruments arecommercially available (such as the Zetasizer Nano Series, MalvernInstruments, Worcestershire, UK). Preferably, DLS is used in the contextof the present invention to determine the polydispersity index (PDI)and/or the main peak diameter of the nanoparticles comprising orconsisting of complexed long-chain RNA molecule, preferably in a liquidcomposition obtained by reconstitution of the inventive dry powder in asuitable solvent, preferably in water, more preferably in water forinjection.

According to a preferred embodiment, the polydispersity index (PDI),preferably the PDI as determined by DLS, of the nanoparticles comprisingor consisting of complexed long-chain RNA molecule is equal to or largerthan 0.10, equal to or larger than 0.11, equal to or larger than 0.12,equal to or larger than 0.13, equal to or larger than 0.14, equal to orlarger than 0.15, equal to or larger than 0.16, equal to or larger than0.17, equal to or larger than 0.18, equal to or larger than 0.19, equalto or larger than 0.20, equal to or larger than 0.21, equal to or largerthan 0.22, equal to or larger than 0.23, equal to or larger than 0.24,equal to or larger than 0.25 or equal to or larger than 0.26. It isparticularly preferred that the PDI, preferably the PDI as determined byDLS, of the nanoparticles comprising or consisting of complexedlong-chain RNA molecule is equal to or larger than 0.12, more preferablyequal to or larger than 0.13, most preferably equal to or larger than0.19 or 0.21. Alternatively, the PDI, preferably the PDI as determinedby DLS, of the nanoparticles comprising or consisting of complexedlong-chain RNA molecule is in a range from 0.10 to 0.40, more preferablyin a range from 0.13 to 0.30, most preferably in a range from 0.19 to0.27.

It is further preferred that the main peak diameter, preferably the mainpeak diameter as determined by DLS, of the nanoparticles comprising orconsisting of complexed long-chain RNA molecule is equal to or largerthan 320 nm, equal to or larger than 325 nm, equal to or larger than 330nm, equal to or larger than 335 nm, equal to or larger than 340 nm,equal to or larger than 345 nm, equal to or larger than 350 nm, equal toor larger than 355 nm, equal to or larger than 360 nm, equal to orlarger than 365 nm, equal to or larger than 370 nm, equal to or largerthan 375 nm, equal to or larger than 380 nm, equal to or larger than 385nm, equal to or larger than 390 nm, equal to or larger than 395 nm orequal to or larger than 400 nm. It is particularly preferred that themain peak diameter, preferably the main peak diameter as determined byDLS, of the nanoparticles comprising or consisting of complexedlong-chain RNA molecule is equal to or larger than 325 nm, morepreferably equal to or larger than 330 nm, most preferably equal to orlarger than 335 nm or 340 nm. Alternatively, the main peak diameter,preferably the main peak diameter as determined by DLS, of thenanoparticles comprising or consisting of complexed long-chain RNAmolecule is in a range from 300 nm to 400 nm, more preferably in a rangefrom 330 nm to 400 nm, most preferably in a range from 327 nm to 390 nm.

In a preferred embodiment, the Zeta potential of the nanoparticlescomprising or consisting of complexed long-chain RNA molecule isdetermined. Methods for determining the Zeta potential are known in theart. For example, the Zeta potential of the nanoparticles comprising orconsisting of complexed long-chain RNA molecule can be determined byusing a Zetasizer Nano Series (Malvern Instruments, Worcestershire, UK).Preferably, the Zeta potential of the nanoparticles comprising orconsisting of complexed long-chain RNA molecule is in a range from −36mV to −50 mV, more preferably from −36 mV to −45 mV.

According to a preferred embodiment, the inventive dry powder isreconstituted in a suitable solvent, preferably in water for injection,and analyzed with respect to the concentration of particles having acertain size, preferably by micro-flow imaging (MFI). For instance,protamine-formulated RNA, which has preferably been spray-dried andreconstituted as described in the Examples 1 to 6 herein, may beanalyzed by MFI as described in Example 8.3.6. In that particularembodiment, the concentration of particles having a diameter of at least1 μm is equal to or less than 175/ml, equal to or less than 170/ml,equal to or less than 165/ml, equal to or less than 160/ml, equal to orless than 155/ml, equal to or less than 150/ml, equal to or less than145/m1 or equal to or less than 140/ml. More preferably, theconcentration of particles having a diameter of at least 2 μm is equalto or less than 35/ml, equal to or less than 32/ml, equal to or lessthan 31/ml, equal to or less than 30/ml, equal to or less than 29/ml,equal to or less than 28/ml, equal to or less than 27/ml, equal to orless than 26/ml or equal to or less than 25/ml.

According to another embodiment, the dry powder composition as describedherein comprises a long-chain RNA, which is preferably not complexedwith poly(lactide-co-glycolide) PLGA. More preferably, the dry powdercomposition as described herein does not comprise PLGA. Alternatively,the dry powder composition as described herein comprises a long-chainRNA, which is preferably not complexed with a compound selected from thegroup consisting of PLGA, poly-lactide (PLA), polyethylene imine (PEI)or poly-L-lysine (PLL). More preferably, the dry powder composition asdescribed herein does not comprise a compound selected from the groupconsisting of PLGA, PLA, PEI or PLL.

According to a further embodiment, the dry powder composition asdescribed herein comprises a long-chain RNA, which is preferably notcomplexed with DOTAP. More preferably, the dry powder composition asdescribed herein does not comprise DOTAP. Even more preferably, the drypowder composition as described herein may comprise a long-chain RNA,which is preferably not complexed with a cationic lipid. Morepreferably, the dry powder composition as described herein does notcomprise a cationic lipid.

In an alternative embodiment, the dry powder composition as describedherein comprises a long-chain RNA, which is preferably not complexedwith mannitol, trehalose or lactose. More preferably, the dry powdercomposition as described herein does not comprise mannitol, trehalose orlactose. Even more preferably, the dry powder composition as describedherein may comprise a long-chain RNA, which is preferably not complexedwith a carbohydrate. More preferably, the dry powder composition asdescribed herein does not comprise a carbohydrate.

In some embodiments, the long-chain RNA of the dry powder composition asdescribed herein is not comprised in nanoparticles or in liposomes,preferably as defined herein. Furthermore, the dry powder compositionmay preferably not comprise a nanoparticle or a liposome, preferably asdefined herein.

In a preferred embodiment, the inventive dry powder compositioncomprising a long-chain RNA molecule comprises at least one furthercomponent or excipient.

In a preferred embodiment, the inventive dry powder compositioncomprises a solvent, preferably in the amounts as defined herein withrespect to the residual moisture content of the dry powder composition.Typically, the solvent is a residue of a solvent, which was used duringpreparation of the dry powder composition, a residue of which may bepresent in the inventive dry powder composition. Preferably, the solventcontained in the inventive dry powder composition is a residue of asolvent used during preparation of the dry powder composition by usingthe inventive method as described herein.

In one embodiment, the solvent comprised in the dry powder compositionaccording to the invention is suitable for use in spray drying.Preferably, a solvent is comprised in the inventive composition, inwhich the long-chain RNA and any other component comprised in thecomposition, if present, are soluble. More preferably, the solvent isvolatile with a boiling point of preferably below 150° C. In addition,the solvent is preferably non-toxic. Preferably, the solvent is anaqueous solution. In the case of an organic solvent, the solvent ispreferably miscible with water.

In a preferred embodiment, the solvent comprised in the dry powdercomposition according to the invention comprises an aequeous solution orwater, preferably pyrogen-free water or water for injection (WFI). Inthis context, the term “water for injection” (WFI) is a term defined bystandard USP 23. USP 23 monograph states that “Water for Injection (WFI)is water purified by distillation or reverse osmosis.” WFI is typicallyproduced by either distillation or 2-stage reverse osmosis. WFItypically does not contain more than 0.25 USP endotoxin units (EU) perml. Endotoxins are a class of pyrogens that are components of the cellwall of Gram-negative bacteria (the most common type of bacteria inwater), preferably in an action limit of 10 cfu/100 ml. The microbialquality may be tested by membrane filtration of a 100 ml sample andplate count agar at an incubation temperature of 30 to 35 degreesCelsius for a 48-hour period. The chemical purity requirements of WFIare typically the same as of PW (purified water).

As a further excipient, the dry powder composition according to theinvention may comprise a buffer, preferably selected from a buffer asdefined herein, e.g. a buffer containing 2-hydroxypropanoic acid,preferably including at least one of its optical isomers L-(+)-lacticacid, (S)-lactic acid, D-(−)-lactic acid or (R)-lactic acid, morepreferably its biologically active optical isomer L-(+)-lactic acid, ora salt or an anion thereof, preferably selected from sodium-lactate,potassium-lactate, or Al₃ ⁺-lactate, NH₄ ⁺-lactate, Fe-lactate,Li-lactate, Mg-lactate, Ca-lactate, Mn-lactate or Ag-lactate, or abuffer selected from Ringer's lactate (RiLa), lactated Ringer's solution(main content sodium lactate, also termed “Hartmann's Solution” in theUK), acetated Ringer's solution, or ortho-lactate-containing solutions(e.g. for injection purposes), or lactate containing water. A buffer asdefined herein may also be a mannose containing buffer, an isotonicbuffer or solution, preferably selected from isotonic saline, a lactateor ortho-lactate-containing isotonic solution, a isotonic buffer orsolution selected from phosphate-buffered saline (PBS), TRIS-bufferedsaline (TBS), Hank's balanced salt solution (HBSS), Earle's balancedsalt solution (EBSS), standard saline citrate (SSC), HEPES-bufferedsaline (HBS), Grey's balanced salt solution (GBSS), or normal saline(NaCl), hypotonic (saline) solutions with addition of glucose ordextrose, or any solution as defined herein, etc. These isotonic buffersor solutions are preferably prepared as defined herein or according toprotocols well known in the art for these specific isotonic buffers orsolutions. In this context, a buffer or, in particular, a residuethereof, may be comprised in the dry powder composition according to theinvention, more preferably an aqueous (isotonic solution or aqueous)buffer, containing a sodium salt, preferably at least 50 mM of a sodiumsalt, a calcium salt, preferably at least 0.01 mM of a calcium salt, andoptionally a potassium salt, preferably at least 3 mM of a potassiumsalt. According to a preferred embodiment, the sodium, calcium and,optionally, potassium salts may occur in the form of their halogenides,e.g. chlorides, iodides, or bromides, in the form of their hydroxides,carbonates, hydrogen carbonates, or sulfates, etc. Without being limitedthereto, examples of sodium salts include e.g. NaCl, NaI, NaBr, Na₂CO₃,NaHCO₃, Na₂SO₄, examples of the optional potassium salts include e.g.KCl, KI, KBr, K₂CO₃, KHCO₃, K₂SO₄, and examples of calcium salts includee.g. CaCl₂, CaI₂, CaBr₂, CaCO₃, CaSO₄, Ca(OH)₂. Typically, the salts arepresent in such a buffer in a concentration of at least 50 mM sodiumchloride (NaCl), at least 3 mM potassium chloride (KCl) and at least0.01 mM calcium chloride (CaCl₂). Furthermore, organic anions of theaforementioned cations may be contained in the buffer. According to amore preferred embodiment, the buffer may contain salts selected fromsodium chloride (NaCl), calcium chloride (CaCl₂) and optionallypotassium chloride (KCl), wherein further anions may be present inaddition to the chlorides. CaCl₂ may also be replaced therein by anothersalt like KCl.

According to a particularly preferred embodiment, the inventive drypowder composition, may be reconstituted in a solvent or a buffer asdefined herein, preferably as defined above. For example, the inventivedry powder composition may be reconstituted in water, Ringer Lactatesolution, a buffer as defined above, or a buffer containing mannose, toobtain the desired salt concentration or alternatively the desiredbuffer conditions. The reconstitution of the dry powder composition iscarried out in WFI (water for injection), if the dry powder compositionwas prepared from a long-chain RNA molecule dissolved in Ringer Lactatesolution (optionally comprising further components), which represents anisotonic solution for injection. In a particularly preferred embodiment,the dry powder composition is reconstituted in an isotonic solution,preferably as defined herein, more preferably in Ringer Lactate,especially if the dry powder composition was prepared from a long-chainRNA molecule dissolved in water, preferably WFI (wherein the wateroptionally comprises further components).

In a preferred embodiment, the dry powder composition according to theinvention does not comprise a lipid compound.

The inventive dry powder composition may further comprise any type ofsuitable component, which is compatible with the long-chain RNAmolecule. As used herein, the term ‘component’ preferably comprises anyadditive or excipient, preferably a pharmaceutically acceptableexcipient that does preferably not cause or enhance degradation of thelong-chain RNA molecule. Such a component may further be in any state,such as liquid, gel-like, solid or semi-solid. A component is preferablyselected from the group consisting of cryoprotectants, lyoprotectants,bulking agents, preservatives, antioxidants, antimicrobial agents,colorants, carriers, fillers, film formers, redispersants anddisintegrants. Moreover, the inventive dry powder composition may alsocomprise excipients, such as defoamers, surfactants, viscosity enhancingagents, force control agents or the like.

Preferably, the inventive dry powder composition comprises at least onecomponent selected from a cryoprotectant, a lyoprotectant or a bulkingagent. In this context, cryoprotectants are understood as excipients,which allow influencing the structure of a frozen material and/or theeutectical temperature of the mixture. Lyoprotectants are typicallyexcipients, which partially or totally replace the hydration spherearound a molecule and thus prevent catalytic and hydrolytic processes. Abulking agent (e.g. a filler) is any excipient compatible with thelong-chain RNA molecule, which may be comprised in the inventivecomposition. As used herein, a bulking agent may be used for increasingthe volume and/or the mass of the inventive composition. In addition, abulking agent may also protect the long-chain RNA molecule fromdegradation.

As a particularly preferred component, the inventive dry powdercomposition may additionally contain at least one suspending agent,preferably mannit.

As a further component, the inventive dry powder composition mayadditionally contain at least one component selected, e.g., fromproteins, amino acids, alcohols, carbohydrates, mannose, mannit, metalsor metal ions, surfactants, polymers or complexing agents, buffers,etc., or a combination thereof.

In the context of the present invention, one preferred component mayalso be selected from the group of amino acids. Such group may comprise,without being limited thereto, any naturally occurring amino acid,including alanine, cysteine, aspartic acid, glutamic acid,phenylalanine, glycine, histidine, isoleucine, lysine, leucine,methionine, asparagine, pyrrolysine, proline, glutamine, arginine,serine, threonine, selenocysteine, valine, tryptophan, and tyrosine,more preferably glycine, arginine, and alanine. Cryoprotectants and/orlyoprotectants selected from the group of amino acids may additionallycomprise any modification of a naturally occurring amino acid as definedabove.

Furthermore, in the context of the present invention, a furthercomponent may be selected from the group of alcohols. Such group maycomprise, without being limited thereto, any alcohol suitable for thepreparation of a pharmaceutical composition, preferably, without beinglimited thereto, mannitol, polyethyleneglycol, polypropyleneglycol,sorbitol, etc.

Additionally, in the context of the present invention, a furthercomponent may be selected from the group of (free) carbohydrates. Ingeneral, a carbohydrate, such as a sugar, can act, for example, as abulking agent, enhance cell targeting (e.g., galactose, lactose), opencellular junctions (e.g., mannitol), and modulate, for instance, thepowder's flowability by altering particle density. Such group of (free)carbohydrates may comprise, without being limited thereto, any (free)carbohydrate, suitable for the preparation of a pharmaceuticalcomposition, preferably, without being limited thereto, (free)monosaccharides, such as e.g. (free) glucose, (free) fructose, (free)galactose, (free) sorbose, (free) mannose (“free” preferably meansunbound or unconjugated, e.g. the mannose is not covalently bound to thelong-chain RNA molecule, or in other words, the mannose is unconjugated,preferably with respect to the long-chain RNA molecule), etc., andmixtures thereof; disaccharides, such as e.g. lactose, maltose, sucrose,trehalose, cellobiose, etc., and mixtures thereof; polysaccharides, suchas raffinose, melezitose, maltodextrins, dextrans, starches, etc., andmixtures thereof; and alditols, such as mannitol, xylitol, maltitol,lactitol, xylitol sorbitol, pyranosyl sorbitol, myoinositol, etc., andmixtures thereof. Examples of sugars that are preferably used in thecomposition according to the invention include lactose, sucrose ortrehalose. Generally, a sugar that is preferred in this context, has ahigh water displacement activity and a high glass transitiontemperature. Furthermore, a sugar suitable for use in the composition ispreferably hydrophilic but not hygroscopic. In addition, the sugarpreferably has a low tendency to crystallize, such as trehalose.Trehalose is particularly preferred.

In an alternative embodiment, the dry powder composition may comprise acryoprotectant, which is preferably not selected from lactose ortrehalose. More preferably, the cryoprotectant is not a carbohydrate.

The weight ratio of the long-chain RNA molecule in the composition tothe carbohydrate component, preferably a sugar, more preferablytrehalose, in the composition is preferably in the range from about1:2.000 to about 1:10, more preferably from about 1:1,000 to about1:100. Most preferably, the weight ratio of the long-chain RNA moleculein the composition to the carbohydrate excipient, preferably a sugar,more preferably trehalose, in the composition is in the range from about1:250 to about 1:10 and more preferably in the range from about 1:100 toabout 1:10 and most preferably in the range from about 1:100 to about1:50.

In preferred embodiment, the dry powder composition according to thepresent invention comprises at least 50% (w/w), preferably at least 70%(w/w), at least 80% (w/w), at least 90% (w/w), or at least 95% (w/w) ofa carbohydrate component, preferably a sugar, more preferably trehalose.

In a particularly preferred embodiment, the inventive dry powdercomposition comprises trehalose. More preferably, trehalose is presentin the inventive dry powder composition in a relative amount of about 5%to about 99.5% (w/w), preferably in a relative amount of about 20% toabout 98% (w/w), more preferably in a relative amount of about 50% toabout 95% (w/w), even more preferably in a relative amount of about 70to about 99% (w/w), and most preferably in a relative amount of about 75to about 90% (w/w). Preferably, the relative amount of trehalose in theinventive dry powder composition is at least 30% (w/w), at least 40%(w/w), at least 50% (w/w), at least 60% (w/w), at least 70% (w/w), atleast 80% (w/w), at least 90% (w/w) or at least 95% (w/w).

In the context of the present invention, a further suitable componentmay also be selected from the group of proteins. Such group maycomprise, without being limited thereto, proteins such as albumin,gelatine, therapeutically active proteins, antibodies, antigens, or anyfurther protein encoded by the long-chain RNA molecule as definedherein.

A component, which may be contained in the inventive dry powdercomposition may be selected from the group of metals or metal ions,typically comprising, without being limited thereto, metals or metalions or salts selected from

alkali metals, including members of group 1 of the periodic table:lithium (Li), sodium (Na), potassium (K), rubidium (Rb), caesium (Cs),and francium (Fr), and their (monovalent) metal alkali metal ions andsalts; preferably lithium (Li), sodium (Na), potassium (K), and their(monovalent) metal alkali metal ions and salts;alkaline earth metals, including members of group 2 of the periodictable: beryllium (Be), magnesium (Mg), calcium (Ca), strontium (Sr),barium (Ba) and radium (Ra), and their (divalent) alkaline earth metalions and salts; preferably magnesium (Mg), calcium (Ca), strontium (Sr),barium (Ba) and their (divalent) alkaline earth metal ions and salts;transition metals, including members of groups 3 to 13 of the periodictable and their metal ions and salts. The transition metals typicallycomprise the 40 chemical elements 21 to 30, 39 to 48, 71 to 80, and 103to 112. The name transition originates from their position in theperiodic table of elements. In each of the four periods in which theyoccur, these elements represent the successive addition of electrons tothe d atomic orbitals of the atoms. In this way, the transition metalsrepresent the transition between subgroup 2 elements and subgroup 12 (or13) elements. Transition metals in the context of the present inventionparticularly comprise members of subgroup 3 of the periodic table:including Scandium (Sc), Yttrium (Y), and Lutetium (Lu), members ofsubgroup 4 of the periodic table: including Titan (Ti), Zirconium (Zr),and Hafnium (Hf), members of subgroup 5 of the periodic table: includingVanadium (V), Niobium (Nb), and Tantalum (Ta), members of subgroup 6 ofthe periodic table: including Chrome (Cr), Molybdenum (Mo), and Tungsten(W), members of subgroup 7 of the periodic table: including Manganese(Mn), Technetium (Tc), and Rhenium (Re), members of subgroup 8 of theperiodic table: including Iron (Fe), Ruthenium (Ru), and Osmium (Os),members of subgroup 9 of the periodic table: including Cobalt (Co),Rhodium (Rh), and Iridium (Ir), members of subgroup 10 of the periodictable: including Nickel (Ni), Palladium (Pd), and Platin (Pt), membersof subgroup 11 of the periodic table: including Copper (Cu), Silver(Ag), and Gold (Au), members of subgroup 12 of the periodic table:including Zinc (Zn), Cadmium (Cd), and Mercury (Hg); preferably membersof period 4 of any of subgroups 1 to 12 of the periodic table: includingScandium (Sc), Titanium (Ti), Vanadium (V), Chromium (Cr), Manganese(Mn), Iron (Fe), Cobalt (Co), Nickel (Ni), Copper (Cu) and Zinc (Zn) andtheir metal ions and salts;earth metals or members of the boron group, including members of group 3of the periodic table: including Boron (B), Aluminium (Al), Gallium(Ga), Indium (In) and Thallium (TI) and their metal ions and salts;preferably Boron (B) and Aluminium (Al) and their metal ions and salts;metalloids or semi metals: including Boron (B), Silicon (Si), Germanium(Ge), Arsenic (As), Antimony (Sb), Tellurium (Te). and Polonium (Po),and their semi metal ions and salts; preferably Boron (B) and Silicon(Si) and their semi metal ions and salts;

In the context of the present invention, a further component may beselected from the group of surfactants may comprise, without beinglimited thereto, any surfactant, preferably any pharmaceuticallyacceptable surfactant, which is preferably suitable for spray drying.More preferably, without being limited thereto, the surfactant isselected from the group consisting of Tween, e.g. Tween 80 (0.2%),Pluronics, e.g. Pluronic L121 (1.25%), Triton-X, SDS, PEG, LTAB,Saponin, Cholate, etc.

As another component, the inventive dry powder composition mayadditionally contain one or more compatible solid or liquid fillers ordiluents or encapsulating compounds, which are preferably suitable foradministration to a patient to be treated. The term “compatible” as usedhere means that these constituents are capable of being mixed with thelong-chain RNA molecule (free or in a complex with a cationic orpolycationic compound), as defined according to the present invention,in such a manner that no interaction occurs, which would substantiallyreduce the integrity or biological activity of the long-chain RNAmolecule, under typical use conditions. Pharmaceutically acceptablecarriers, fillers and diluents must, of course, have sufficiently highpurity and sufficiently low toxicity to make them suitable foradministration to a person to be treated. Some examples of compounds,which can be used as pharmaceutically acceptable carriers, fillers orconstituents thereof are sugars, such as, for example, lactose, glucoseand sucrose; starches, such as, for example, corn starch or potatostarch; cellulose and its derivatives, such as, for example, sodiumcarboxymethylcellulose, ethylcellulose, cellulose acetate; powderedtragacanth; malt; gelatin; tallow; solid glidants, such as, for example,stearic acid, magnesium stearate; calcium sulfate; vegetable oils, suchas, for example, groundnut oil, cottonseed oil, sesame oil, olive oil,corn oil and oil from theobroma; polyols, such as, for example,polypropylene glycol, glycerol, sorbitol, mannitol and polyethyleneglycol; alginic acid.

In addition, the dry powder composition according to the invention mayoptionally contain further excipients or agents, such as stabilizers,for example EDTA, Tween, benzoic acid derivatives or RNAse inhibitors.Preferably, the dry powder composition may further comprise any type ofcomponent or additive, which is compatible with the long-chain RNAmolecule. Such an excipient is preferably selected from the groupconsisting of preservatives, antioxidants, antimicrobial agents,colorants, carriers, fillers, film formers, redispersants anddisintegrants. Moreover, the dry powder composition may also comprise acomponent or additive, preferably in very small amounts, that were addedduring the manufacturing process, such as defoamers, surfactants,viscosity enhancing agents, force control agents or the like.

In a preferred embodiment, the dry powder composition of the inventionis obtained by the method as described herein.

As explained herein, the dry powder composition according to theinvention is particularly suitable as storage-stable form of along-chain RNA molecule. The inventors have surprisingly found that thestorage stability of the long-chain RNA molecule in the dry powdercomposition is excellent and the long-chain RNA molecule remainsfunctional after extended storage periods. The storage stability of thelong-chain RNA molecule is typically determined through determination ofthe relative (structural) integrity and the biological activity after agiven storage period, e.g. via time-course in vitro expression studies.

The relative integrity is preferably determined as the percentage offull-length RNA (i.e. non-degraded long-chain RNA) with respect to thetotal amount of RNA (i.e. long-chain RNA and degraded RNA fragments(which appear as smears in gel electrophoresis)), preferably afterdeduction of the LOD (3×background noise), for example, by using thesoftware QuantityOne from BioRad.

The dry powder composition according to the invention thus provides theadvantageous characteristics of a powder and the potential of such acomposition for, e.g. packaging and dosage, while it also allowssignificantly longer storage at temperatures from −80° C. to 60° C. thanthe corresponding RNAs in WFI or other injectable solutions.Particularly, it can be stored at room temperature, which simplifiesshipping and storage. Preferably, the dry powder composition is storedwith or without shielding gas. In one embodiment, single doses of thedry powder composition are packaged and sealed. Alternatively, multipledoses can be packaged in one packaging unit. Single dose packaging inblisters or capsules is preferably used in order to preventcross-contamination.

Preferably, the relative integrity is at least 70%, more preferably atleast 75%, at least 80%, at least 85%, at least 90% or at least 95%after storage at room temperature for preferably at least one week, morepreferably for at least one month, even more preferably for at least 6months and most preferably for at least one year.

Further preferably, the biological activity of the long-chain RNAmolecule of the dry powder composition after storage at roomtemperature, preferably as defined above with respect to the relativeintegrity of the long-chain RNA molecule, is preferably at least 70%,more preferably at least 75%, at least 80%, at least 85%, at least 90%or at least 95% of the biological activity of the freshly preparedlong-chain RNA molecule. The biological activity is preferablydetermined by analysis of the amounts of protein expressed fromreconstituted RNA and from freshly prepared RNA, respectively, e.g.after transfection into a mammalian cell line. Alternatively, thebiological activity may be determined by measuring the induction of an(adaptive or innate) immune response in a subject.

In a further aspect of the invention, a method is provided that allowspreparing a storage form of a long-chain RNA molecule, preferably along-chain RNA in particulate form as described herein, wherein a liquidcomprising the RNA molecule is provided and wherein the liquid is driedby spray-drying. In one embodiment, the invention concerns a method fordrying a liquid comprising a long-chain RNA molecule.

With respect to the following description of the inventive method, it isnoted that the definitions and specifications provided above withrespect to the inventive dry powder composition may likewise apply tothe inventive method. In particular, the description of the long-chainRNA molecule and further components of the dry powder composition applyto the inventive method as well. In addition, further definitions mayapply to the inventive method as specifically indicated in thefollowing.

In a preferred embodiment, the invention concerns a method for preparinga dry powder comprising a long-chain RNA molecule, wherein the methodcomprises the following steps:

a) providing a liquid comprising the long-chain RNA molecule,b) drying the liquid provided in step a) by spray-drying.

The inventors found that a storage form of a long-chain RNA molecule maybe obtained by the method as described herein. In particular, theinventors found that—by using the method according to the invention—adry powder comprising a long-chain RNA molecule can be obtained.Advantageously, the method according to the invention is suitable forapplication at an industrial scale. In addition, the invention providesa method that can be carried out by the skilled person using standardequipment, thus providing a cost- and time-effective solution. Moreover,the method can be carried out in bulk as well as continuously. Thestorage form, preferably the long-chain RNA in particulate form,obtained by using the method according to the invention thereforerepresents an effective means for extending the stability of long-chainRNA as an API (active pharmaceutical ingredient), especially duringstorage at a variety of different temperatures and in differentpackaging formats.

In step a) of the method according to the invention, a liquid isprovided that comprises a long-chain RNA molecule. The long-chain RNAmolecule comprised in the liquid provided in step a) of the inventivemethod is characterized by any feature or any combination of featuresdescribed herein with respect to the long-chain RNA molecule that iscomprised in the inventive dry powder composition.

Typically, the liquid in step a) of the method according to theinvention is provided by diluting or dissolving the long-chain RNAmolecule in a suitable solvent. The solvent is preferably a solventsuitable for use in spray drying. Preferably, a solvent is used, inwhich the long-chain RNA and any other component, if present, aresoluble. Suitable solvents are described above with respect to theinventive dry powder composition. The liquid in step a) of the inventivemethod preferably comprises the long-chain RNA molecule and a solvent orbuffer as described above with respect to the inventive dry powdercomposition. Preferably, step a) of the inventive method comprisesdissolving or diluting the long-chain RNA molecule as defined herein ina solvent or buffer as defined herein, preferably in an aqueoussolution, such as Ringer Lactate, or water, more preferably pyrogen-freewater or WFI.

In a further preferred embodiment, the liquid comprising the long-chainRNA molecule comprises at least one further component, preferably asdescribed herein with respect to the dry powder composition disclosedherein. In particular, the liquid provided in step a) of the inventivemethod preferably comprises a further component selected from the groupconsisting of buffers, cryoprotectants, lyoprotectants, bulking agents,suspending agents, proteins, amino acids, alcohols, carbohydrates,metals, metal ions, salts, surfactants, fillers, diluents, carriers,glidants, vegetable oils, polyols, encapsulating compounds, stabilizers,preservatives, antioxidants, antimicrobial agents, colorants, filmformers, redispersants, disintegrants, defoamers, viscosity enhancingagents and force control agents, wherein the respective component ispreferably as defined above with respect to the inventive dry powdercomposition.

The long-chain RNA molecule as defined herein may be present in theliquid provided in step a) of the inventive method in free form (as“naked RNA”) and/or as a complex with a polycationic or cationiccompound, preferably as described herein. The long-chain RNA molecule asdefined herein and a cationic or polycationic compound may be comprisedin the liquid provided in step a), either as a complex, preferably inthe form of a nanoparticle as defined herein, or both in free form, i.e.in solution without being in a complex with each other. The preparationof RNA complexes with complexation agents, preferably with polycationicor cationic compounds, is known in the art and is preferably carried outas described in WO2010/037539 or WO2011/026641, the entire disclosure ofwhich is herewith incorporated by reference.

In a particularly preferred embodiment, the liquid provided in step a)of the inventive method comprises a complexation agent, preferably asdefined herein, more preferably a cationic or polycationic compound asdefined herein, such as protamine, nucleoline, spermin, spermidine,oligoarginines as defined above, such as Arg₇, Arg₈, Arg₉, Arg₇, H₃R₉,R₉H₃, H₃R₉H₃, YSSR₉SSY, (RKH)₄, Y(RKH)₂R, etc. The complexation agent,preferably a cationic or polycationic compound as defined herein, ispreferably present in the liquid provided in step a) in free form (insolution) or in a complex with the long-chain RNA molecule. Protamine isparticularly preferred and is preferably comprised in the liquidprovided in step a) of the method at a concentration in a range from0.01 g/l to 10 g/l, from 0.05 g/l to 5 g/l, or from 0.05 g/l to 2 g/l.More preferably, the concentration of protamine in the liquid providedin step a) of the method is in a range from 0.05 g/l to 3 g/l or from0.1 to 1 g/l.

In a preferred embodiment, the liquid provided in step a) furthercomprises lactate, wherein the lactate concentration is preferably inthe range of about 3 mM to about 300 mM, preferably in the range ofabout 5 mM to about 200 mM, more preferably in the range of about 10 mMto about 150 mM, even more preferably about 15 mM to about 35 mM, andmost preferably 20 mM to about 31 mM. Alternatively, the liquid providedin step a) of the method typically comprises a Ringer's lactateconcentration (or a concentration of any of the afore mentioned lactatecontaining solutions) e.g. in the range of about 10% (w/w) to about 100%(w/w), e.g. in the range of about 20% (w/w) to about 100% (w/w), in therange of about 30% (w/w) to about 100% (w/w), in the range of about 40%(w/w) to about 100% (w/w), in the range of about 50% (w/w) to about 90%(w/w), preferably in the range of about 60% (w/w) to about 90% (w/w),more preferably in the range of about 70% (w/w) to about 90% (w/w), e.g.about 80% (w/w), of Ringer's lactate (or the afore mentioned lactatecontaining solution). In this context, Ringer's lactate (100% (w/w)) istypically defined as a solution comprising 131 mM Na⁺, 5.36 mM K⁺, 1.84mM Ca²⁺, and 28.3 mM Lactate).

In another embodiment, the liquid provided in step a) of the inventivemethod does not comprise a lipid compound.

As a particularly preferred component, the liquid provided in step a) ofthe method may additionally contain at least one suspending agent,preferably mannit, preferably in a concentration of about 1 to 15%(w/w), more preferably in a concentration of about 3 to 10% (w/w), andeven more preferably in a concentration of about 4 to 6% (w/w).

In a further embodiment, the liquid provided in step a) of the methodcomprises a carbohydrate component, preferably a sugar, more preferablytrehalose. In a preferred embodiment, a carbohydrate component,preferably a sugar, more preferably trehalose is present in the liquidprovided in step a) of the method at a concentration of about 0.01 toabout 20% (w/w), preferably in a concentration of about 0.01 to about15% (w/w), more preferably in a concentration of about 0.1 to about 10%(w/w), even more preferably in a concentration of about 0.5 to about 10%(w/w), and most preferably in a concentration of about 2.5 to about 7.5%(w/w), e.g. at a concentration of about 4 to about 7% (w/w), such asabout 5% (w/w).

The pH of the liquid provided in step a) of the method may be in therange of about 4 to 8, preferably in the range of about 6 to about 8,more preferably from about 7 to about 8.

Preferably, the liquid provided in step a) of the method contains theherein defined contents, optional components, additives, etc. in such aconcentration so as to lead to an osmolarity comparable to that of bloodplasma. In this context, the term “osmolarity” is typically to beunderstood as a measure of all contents, optional components, additives,etc. of the liquid as defined herein. More precisely, osmolarity istypically the measure of solute concentration, defined as the number ofosmoles (Osm) of all solubilized contents, optional components,additives, etc. per liter (l) of solution (osmol/l or osm/l). In thepresent context, the liquid provided in step a) of the method maycomprise an osmolarity preferably in the range of about 200 mosmol/l toabout 400 mosmol/l, more preferably in the range of about 250 mosmol/lto about 350 mosmol/l, even more preferably in the range of about 270mosmol/l to about 330 mosmol/l or in the range of about 280 mosmol/l toabout 320 mosmol/l, or in the range of about e.g. about 290 mosmol/l toabout 310 mosmol/l, e.g. about 295 mosmol/l, about mosmol/l, about 296mosmol/l, about 297 mosmol/l, about 298 mosmol/l, about 299 mosmol/l,about, 300 mosmol/l, about 301 mosmol/l, about 302 mosmol/l, about 303mosmol/l, about 304 mosmol/l, about 305 mosmol/l, about 306 mosmol/l,about 307 mosmol/l, about 308 mosmol/l.

The method according to the present invention further comprises a stepb), wherein the liquid provided in step a) of the method is dried byspray-drying.

As used herein, the term ‘spray-drying’ typically relates to a processthat involves breaking up a liquid into small droplets (atomization) andrapidly removing solvent from the droplets in a spray-drying apparatus,where there is a strong driving force for evaporation of solvent fromthe droplets, which provide a favourable surface to mass ratio. Thestrong driving force for solvent evaporation is generally provided by ahigh surface to mass ratio of the droplets and by maintaining thepartial pressure of solvent in the spray-drying apparatus well below thevapor pressure of the solvent at the temperature of the drying droplets.This may be achieved, for example, by maintaining the pressure in thespray-drying apparatus at a partial vacuum or by mixing the dropletswith a warm drying gas or a combination of both. As a result of thespray-drying process, particles, preferably dry particles, morepreferably in the form of a dry powder composition, are typicallyobtained.

The method according to the invention may be carried out in bulk or as acontinuous process. In a preferred embodiment, the method is carried outas a continuous process. In particular, the spray-drying process may becarried out in bulk or as a continuous process in the context of theinventive method. Most preferably, the spray-drying process is carriedout in a continuous process.

Preferably, the liquid provided in step a) of the method is used asliquid feed in a spray-drying process.

Typically, the liquid comprising the long-chain RNA molecule, which isprovided in step a), is first broken up into a plurality of smalldroplets that are preferably suspended in a gas or a gas mixture, suchas air. The obtained mixture of droplets and gas is typically referredto as ‘spray’ or ‘fog’. The process of breaking up the liquid feed intodroplets is known as ‘atomization’ and may be carried out using anysuitable device known in the art (atomizer). Various types of atomizersare known in the art, which are suitable for being used in the inventivemethod, such as rotary atomizers, pressure nozzles, two-fluid nozzles,fountain nozzles, ultrasonic nebulizers and vibrating orifice aerosolgenerators.

In one embodiment, a rotary atomizer is used as atomizer in thespray-drying. Rotary atomizers exploit the energy of high-speed rotationto produce fine droplets. The liquid feed is introduced into areservoir, typically in the center of the rotary wheel.

According to a preferred embodiment, a two-fluid nozzle is used in thespray-drying process. Two-fluid nozzles combine two fluids, where onefluid is typically the liquid feed to be dried and the second fluid istypically a compressed gas (e.g. air, nitrogen or CO₂ at, for example,0.1 to 7 bar). The energy of the compressed gas is used to atomize theliquid feed. Once the liquid feed is atomized, the produced spraydroplets are usually mixed with a drying gas stream, allowing the liquidto quickly evaporate. The rapid evaporation typically results in acooling effect, so that the dried particles do not reach the drying airtemperature, which is particularly advantageous if heat sensitivematerial is dried.

In a preferred embodiment, a pressure nozzle is used as atomizer.Preferably, a pressure nozzle is used, which comprises a swirl chamber,causing the liquid passing through them to rotate. Preferably, apressure nozzle is used as atomizer, wherein the nozzle pressure ispreferably not higher than about 1 bar, more preferably not higher thanabout 0.7 bar, not higher than about 0.5 bar or not higher than about0.3 bar. Preferably, the nozzle pressure is in a range from about 1 toabout 0.1 bar, more preferably in a range from about 0.7 to about 0.3bar. In a particularly preferred embodiment, the nozzle pressure is nothigher than about 0.3 bar.

In the context of the present invention, an atomizer is preferably usedthat produces droplets, which are preferably characterized by a massmedian aerodynamic diameter (MMAD), preferably as defined herein, of atleast 0.3 μm. Alternatively, the MMAD of the droplets according to theinvention is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45 or 50 μm. Preferably, the MMAD of the droplets is equal to orless than 1,500, 1,250, 1,000, 750, 600, 500, 400, 300, 200 or 100 μm.Further preferably, the MMAD of the droplets may be in a range from 0.5μm to 2,000 μm, from 1 μm to 1,000 μm, from 2 μm to 500 μm or from 2 μmto 200 μm. In a preferred embodiment, the MMAD of the droplets is atleast 1 μm or in the range from 1 to 200 μm. In a particularly preferredembodiment, the MMAD of the droplets is at least 3 μm, at least 5 μm orat least 20 μm.

Preferably, the droplet size distribution is narrow, i.e. the size ofthe individual droplets that are formed by the atomizer is relativelyuniform. More preferably, the droplets formed by the atomizer arecharacterized by using the span of the droplet size distribution as aparameter. Therein, the span (for a volume weighted distribution) isdefined as outlined above with respect to the particle size of theinventive dry powder composition. In a preferred embodiment, the dropletsize distribution is characterized by a low span value, which preferablyresults in a narrow particle size distribution in the dry powdercomposition. Typically, a narrow droplet size distribution afteratomization results in increased flowability of the resulting drypowder. Preferably, the span of the droplets formed by the atomizer isequal to or less than 5, more preferably equal to or less than 4, andeven more preferably equal to or less than 3. In a particularlypreferred embodiment, the particle size distribution of the dry powdercomposition according to the invention is characterized by a span ofless than about 2 or less than about 1.5.

In a preferred embodiment, atomization of the liquid feed results inspherical droplets. As used herein, the term “spherical” comprises notonly geometrically perfect spheres, but also more irregular shapes, suchas spheroidal, elipsoid, oval or rounded droplet. Waddell's sphericity ψ(herein also referred to as “sphericity” or “circularity”) may becalculated, e.g. by using the following equation

$\psi = \frac{{surface}\mspace{14mu} {area}{\; \;}{of}\mspace{14mu} {sphere}\mspace{14mu} {of}\mspace{14mu} {equal}\mspace{14mu} {volume}\mspace{14mu} {to}\mspace{14mu} {the}\mspace{14mu} {droplet}}{{surface}{\mspace{11mu} \;}{area}{\mspace{11mu} \;}{of}{\mspace{11mu} \;}{the}{\; \mspace{11mu}}{droplet}}$

In a preferred embodiment, a droplet formed by atomization of the liquidfeed is characterized by a sphericity of at least 0.7, preferably atleast 0.75, at least 0.8, at least 0.85, at least 0.9, at least 0.95or 1. Preferably, the atomizer generates a plurality of dropletscomprising at least one droplet with a sphericity in the range from 0.7to 1, more preferably in the range from 0.8 to 1, from 0.85 to 1, orfrom 0.9 to 1.

It is further preferred that the average sphericity of the droplets,which are formed by the atomizer, is at least 0.7, preferably at least0.75, at least 0.8, at least 0.85, at least 0.9, at least 0.95 or 1.Preferably, the average sphericity of the droplets, which are formed bythe atomizer, is in the range from 0.7 to 1, more preferably in therange from 0.8 to 1, from 0.85 to 1, or from 0.9 to 1.

Alternatively, the sphericity of those droplets that have a particlesize (i.e. droplet size) equal to Dv50 in the drople size distributionas defined herein is at least 0.7, preferably at least 0.75, at least0.8, at least 0.85, at least 0.9, at least 0.95 or 1. Preferably, thesphericity of those droplets that have a particle size equal to Dv50 asdefined herein is in the range from 0.7 to 1, more preferably in therange from 0.8 to 1, from 0.85 to 1, or from 0.9 to 1. Even morepreferably, those droplets that have a particle size equal to Dv90 asdefined herein have a sphericity of at least 0.7, preferably of at least0.75, at least 0.8, at least 0.85, at least 0.9, at least 0.95 or 1.Preferably, the sphericity of those droplets that have a particle sizeequal to Dv90 as defined herein is in the range from 0.7 to 1, morepreferably in the range from 0.8 to 1, from 0.85 to 1, or from 0.9 to 1.

Further preferably, the average sphericity of those droplets that have aparticle size equal to or lower than Dv50 as defined herein is at least0.7, preferably at least 0.75, at least 0.8, at least 0.85, at least0.9, at least 0.95 or 1. Preferably, the average sphericity of thosedroplets that have a particle size equal to or lower than Dv50 asdefined herein is in the range from 0.7 to 1, more preferably in therange from 0.8 to 1, from 0.85 to 1, or from 0.9 to 1. Even morepreferably, the average sphericity of those droplets that have aparticle size equal to or lower than Dv90 as defined herein is at least0.7, preferably of at least 0.75, at least 0.8, at least 0.85, at least0.9, at least 0.95 or 1. Preferably, the average sphericity of thosedroplets that have a particle size equal to or lower than Dv90 asdefined herein is in the range from 0.7 to 1, more preferably in therange from 0.8 to 1, from 0.85 to 1, or from 0.9 to 1.

In a preferred embodiment, the droplets formed by the atomizer are driedby evaporation of the solvent from the droplets. Typically, this takesplace in a drying chamber, which may be of any shape and which mayconsist of one or more chambers. Preferably, the droplets are contactedwith a gas stream that is preferably capable of absorbing, at leastpartially, the solvent that evaporates from the droplets. The gas streamis preferably introduced into the drying chamber via an inlet, such as adisperser, which is preferably located in the upper half of the dryingchamber, more preferably in the vicinity of the atomizer, thus allowingrapid mixing of the drying gas and the droplets. The gas stream leavesthe drying chamber through an outlet, which is preferably located at thebottom of the drying chamber. In a preferred embodiment, the dryingchamber comprises a cone-shaped part, wherein the tip of the conecomprises the outlet, preferably for the drying gas as well as for driedparticles.

Preferably, the characteristics of the drying chamber are matched with,amongst others, the atomizer that is used. In order to ensure uniformproduct quality, the droplets preferably contact a surface only whenthey are sufficiently dry. Spray-drying devices that use centrifugalatomizer typically require relatively larger diameter vessels, but lesscylinder height for optimal drying. Spray-drying devices that usepressure nozzles usually require relatively small diameters with largercylinder height for sufficient drying. The dry powder composition ispreferably collected at the bottom of the drying chamber that ispreferably designed as a cone. In the center of the cone area, theoutlet of the gas-stream is preferably positioned, where cool and moistair is removed from the drying chamber. Such a design of the cone andoutlet is acting as a cyclone separator and leads to an accumulation ofthe dry powder composition at the bottom of the drying chamber. Cyclonicseparation is preferably used to separate dry particles or fine dropletsfrom the drying gas, preferably without the use of filters, throughvortex separation. To this end, a high speed rotating flow is preferablyestablished within a cylindrical or conical container, the cyclone.Typically, drying gas flows in a helical pattern, from the top (wideend) of the cyclone to the bottom (narrow) end before exiting thecyclone in a straight stream through the center of the cyclone and outthe top. Larger or denser particles in the rotating stream do not followthe tight curve of the stream, but strike the outside wall and fall tothe bottom of the cyclone, where they can be collected. Alternatively, afilter, e.g. a bag filter or a combination of a cyclone separator and afilter may be used for separation.

Depending on the type of flow, i.e. the relative positions of atomizerand drying gas inlet or, respectively, the relative movement of thespray and the drying gas, several types of spray-drying devices may bedistinguished, all of which may be used in the method according to theinvention. In a preferred embodiment the spray-drying device is set upas a co-current flow device (spray and drying gas move into the samedirections), as a counter-current flow device (spray and drying gas moveinto opposite directions) or as a mixed flow device (co-current andcounter-current flow combined). In a particularly preferred embodiment,the spray-drying device is a co-current flow device.

Moreover, common spray-drying devices may be categorized depending onthe type of air cycle that is used. A spray-drying device as used hereinis preferably an open cycle device (drying air that enters the systemthrough the inlet is exhausted through the outlet into the atmosphere)or a closed cycle device (drying air that enters the system through theinlet, exits the system via the outlet and is reused). A closed cycledevice is preferably employed, if a drying gas is used that should notaccumulate in the facility, where the device is located (such asnitrogen or CO₂), and/or if toxic substances are released from thedrying material.

The spray-drying device preferably reduces the residual moisture contentof the composition to the desired level, preferably as defined herein,in one pass through the system. If the moisture content of the productafter one cycle is higher than desired, the moisture content of thepowder may be further reduced by a second drying stage (or several ofthose) until the desired residual moisture content of the product isachieved.

An example of a spray-drying apparatus is shown in FIG. 1, which furtherillustrates the principle of spray-drying. Liquid input stream (A) issprayed through a nozzle (3) into a hot vapor stream (1, 2) and isvaporized (4). Upon introduction into the hot air stream the dropletsare cooled down due to the evaporation of water or a chemical solventfrom the concentrate. Solid particles form, while moisture quicklyleaves the droplets. In the example illustrated in FIG. 3, a nozzle isused in order to achieve a sufficiently small droplet size (atomizer)and in order to maximize heat transfer and the rate of water/solventevaporation. A co-current flow of hot drying gas is used in theexemplary device shown in FIG. 3. Particles are further dried andseparated in a cyclone device (6). The dry particles are cooled andcollected, ready for packaging in different formats (8).

The final product is collected as described above and is preferably inthe form of a dry powder comprising the particles as defined herein withrespect to the inventive dry powder composition.

The spray-drying process, in the context of the present invention, maybe carried out using any suitable spray-drying device known in the art.Examples of commercially available devices that may be employed include,but are not limited to the following examples: Mini Spray Dryer B-290(Buchi); Nano Spray Dryer B-90 (Buchi); Anhydro MicraSpray Dryer GMP(SPX.com); Anhydro MicraSpray Dryer Aseptic series (SPX.com); MDL-50series; B,C,S,M sub-types. (fujisaki electric); MDL-015 (C) MGClab-scale. (fujisaki electric); MDL-050 (C) MGC lab-scale. (fujisakielectric); LSD-1500 Mini spray dryer (cndryer.com); MSD-8Multi-functional laboratory spray dryer (cndryer.com); PSD-12 Precisionpharmacy spray dryer (cndryer.com); PSD-12 Precision pharmacy spraydryer (cndryer.com); TALL FORM DRYER™—TFD (GEA Process Engineering);COMPACT DRYER™—CD (GEA Process Engineering); Multi-Stage Dryer—MSD™ (GEAProcess Engineering); FILTERMAT™ Spray Dryer—FMD (GEA ProcessEngineering); SDMICRO™ (GEA Process Engineering), MOBILE MINOR™ (GEAProcess Engineering); PRODUCTION MINOR™ (GEA Process Engineering);VERSATILE-SD™ (GEA Process Engineering); FSD™ Fluidized Spray Dryer (GEAProcess Engineering); Standard GEA Niro PHARMASD™ spray dryers (GEAProcess Engineering); R&D Spray Dryer—SDMICRO™ (GEA ProcessEngineering).

In the spray-drying process according to the invention, the drying gasmay be any suitable gas or mixture of gases, such as air. Preferably, aninert gas is used as drying gas, for example nitrogen, nitrogen-enrichedair, helium or argon.

The spray-drying process is influenced to a considerable degree by thetemperature of the drying gas. That temperature is typicallycharacterized by two parameters, i.e. the inlet temperature (T_(inlet))and the outlet temperature (T_(outlet)). As used herein, the term ‘inlettemperature’ refers to the drying gas temperature as measured at thedrying gas inlet of the drying chamber as described herein. Analogously,the term ‘outlet temperature’ refers to the drying gas temperature asmeasured at the drying gas outlet of the drying chamber as describedherein.

For heat sensitive material, the inlet temperature is preferably chosensufficiently high in order to allow rapid and efficient drying, while atthe same time avoiding degradation of the material. If heat sensitivematerial is dried, the residence time of the material in the dryingchamber is preferably increased, e.g. by using larger drying chambers,which allows operating at lower temperatures.

In a preferred embodiment, the outlet temperature is at least about 65°C., at least about 66° C., at least about 67° C., at least about 68° C.,at least about 69° C., at least about 70° C., at least about 71° C., atleast about 72° C., at least about 73° C., at least about 74° C., atleast about 75° C., at least about 76° C., at least about 77° C., atleast about 78° C., at least about 79° C., at least about 80° C., atleast about 85° C., at least about 86° C., at least about 87° C., atleast about 88° C., at least about 89° C., at least about 90° C., atleast about 91° C., at least about 92° C., at least about 93° C., atleast about 94° C., at least about 95° C., at least about 96° C., atleast about 97° C., at least about 98° C., at least about 99° C., atleast about 100° C., at least about 101° C., at least about 102° C., atleast about 103° C., at least about 104° C., at least about 105° C., atleast about 106° C., at least about 107° C., at least about 108° C., atleast about 109° C., or at least about 110° C.

According to a particularly preferred embodiment, the outlet temperatureis at least about 65° C. Alternatively, the outlet temperature is atleast about 69° C. More preferably, the outlet temperature is at leastabout 70° C., most preferably at least about 71° C. or at least about72° C.

Typically, higher temperatures increase the efficiency of thespray-drying process. The disadvantage of higher temperatures may be,however, an increased risk of degradation depending on the activeingredient or the excipients. Another caveat with spray-drying ingeneral are shear forces that are acting on the liquid to be dried, inparticular during atomization. The shear stress is typically expected tobe particularly detrimental to larger molecules. Before the presentinvention, it was therefore altogether unexpected that a long-chain RNAmolecule may be dried by spray-drying, while the long-chain RNAmolecule's integrity and biological activity is retained notwithstandingthe mechanical stress involved in the process. Even more surprisingly,it has been found by the inventors of the present invention that thelong-chain RNA molecule is not degraded, even when relatively hightemperatures, in particular outlet temperatures, are used. In contrast,the skilled person would have expected that high outlet temperatures(T_(outlet)) used in the spray-drying process (for example,T_(outlet)>60° C.) would result in degradation of long-chain RNA. Thisallows the preparation of a particulate, storage-stable form oflong-chain RNA, which advantageously has an outstandingly low residualmoisture content that allows for long-term storage without degradation.

One particular advantage of the inventive dry powder composition and theinventive method is that a dry powder composition is provided, which canbe divided into packages useful for shipping, storage and use asmedicament. Furthermore dry powder formation of long-chain RNArepresents a cost- and time effective process, which can readily bescaled-up for commercial production. In this context, it is particularlyadvantageous that spray drying can be carried out as a continuousprocess. One advantage of a continuous process is that the productproduced in one run has the same properties, therefore reducing theamount of required quality controls.

Preferably, the residual moisture content of the dry powder compositionobtained by the method according to the invention is as defined abovewith regard to the inventive dry powder composition.

More preferably, the relative integrity and the biological activity ofthe long-chain RNA molecule in the dry powder composition obtained byusing the inventive method is preferably as defined above for theinventive dry powder composition comprising a long-chain RNA molecule.

The inventive method thus provides long-chain RNA as defined herein in aparticulate formulation. In a particularly preferred embodiment, theparticles comprised in the dry powder composition obtained by theinventive method are characterized by a size distribution, which ispreferably as defined herein for the particles of the inventive drypowder composition. In a particularly preferred embodiment, the product,which is obtained from the method according to the invention, is theinventive dry powder composition as described herein.

In one aspect, the invention concerns a particle, or a plurality ofparticles, comprising a long-chain RNA molecule, which is preferablyobtainable by the inventive method. Furthermore, the invention isdirected to a dry powder composition comprising a long-chain RNAmolecule, which is obtainable by the inventive method as defined herein.

In a preferred embodiment, the inventive dry powder composition, theparticle obtainable by the inventive method or the dry powdercomposition obtainable by the inventive method are packaged in singledosages after the drying process is completed. Alternatively, the methodmay further comprise purification or selection steps, for example inorder to separate particles of a certain size or shape.

Advantageously, the inventive dry powder composition, the particleobtainable by the inventive method or the dry powder compositionobtainable by the inventive method may also be extended at any stageafter the production process per se is finished. For instance, anexcipient, preferably as described herein, may be added to the inventivedry powder composition or to the product of the inventive method,respectively. In that manner, the product of the inventive methodprovides considerable flexibility and allows extension of weight/volume(e.g. for better handling) as well as combination with other activeingredients and excipients. For example, a suitable excipient,preferably as defined herein, such as a carbohydrate, may be added, forexample inulin, starch or trehalose. Preferably, the excipient, which isadded to inventive dry powder composition or to the particles or drypowder composition obtained by the inventive method, is characterized bya low osmolarity.

If the inventive dry powder composition, the particles or the dry powdercomposition obtainable by the inventive method is used in themanufacture of a pharmaceutical composition, the powder or the particlescan easily be further processed to other dosage forms, such as tablets,capsules, granules or the like.

In a further aspect, the present invention provides a pharmaceuticalcomposition, comprising or consisting of the inventive dry powdercomposition, the particles as obtainable by the inventive method or thedry powder composition obtainable by the inventive method. In apreferred embodiment, the inventive dry powder composition, theparticles as obtainable by the inventive method or the dry powdercomposition obtainable by the inventive method are pharmaceuticalcompositions. Alternatively, the inventive pharmaceutical compositioncomprises the inventive dry powder composition, the particles asobtainable by the inventive method or the dry powder compositionobtainable by the inventive method and optionally a pharmaceuticallyacceptable carrier and/or vehicle. The inventive pharmaceuticalcomposition may optionally be supplemented with further components asdefined above for the inventive dry powder composition or for theinventive method. The inventive pharmaceutical composition may beprepared as a whole by the inventive method.

As a first ingredient, the inventive pharmaceutical compositioncomprises the long-chain RNA in particulate form as defined herein. Inparticular, the first ingredient of the inventive pharmaceuticalcomposition is the inventive dry powder composition, the particles asobtainable by the inventive method or the dry powder compositionobtainable by the inventive method, as defined above. Preferably, thelong-chain RNA molecule as defined herein represents a pharmaceuticallyactive ingredient of the pharmaceutical composition.

As a second ingredient the inventive pharmaceutical composition maycomprise another class of compounds, which may be added to the inventivepharmaceutical composition in this context, may be selected from atleast one pharmaceutically active component. A pharmaceutically activecomponent in this context is a compound that has a therapeutic effectagainst a particular medical indication, preferably cancer diseases,autoimmune disease, allergies, infectious diseases or a further diseaseas defined herein. Such compounds include, without implying anylimitation, preferably compounds including, without implying anylimitation, peptides or proteins (e.g. as defined herein), nucleic acidmolecules, (therapeutically active) low molecular weight organic orinorganic compounds (molecular weight less than 5,000, preferably lessthan 1,000), sugars, antigens or antibodies (e.g. as defined herein),therapeutic agents already known in the prior art, antigenic cells,antigenic cellular fragments, cellular fractions; modified, attenuatedor de-activated (e.g. chemically or by irridation) pathogens (virus,bacteria etc.), etc.

Furthermore, the inventive pharmaceutical composition may comprise apharmaceutically acceptable carrier and/or vehicle. In the context ofthe present invention, a pharmaceutically acceptable carrier typicallyincludes the liquid or non-liquid basis of the inventive pharmaceuticalcomposition. If the inventive pharmaceutical composition is provided inliquid form, the carrier will typically be pyrogen-free water; isotonicsaline or buffered (aqueous) solutions, e.g phosphate, citrate etc.buffered solutions. Particularly for injection of the inventivepharmaceutical composition, water or preferably a buffer, morepreferably an aqueous buffer, may be used, containing a sodium salt,preferably at least 50 mM of a sodium salt, a calcium salt, preferablyat least 0.01 mM of a calcium salt, and optionally a potassium salt,preferably at least 3 mM of a potassium salt. According to a preferredaspect, the sodium, calcium and, optionally, potassium salts may occurin the form of their halogenides, e.g. chlorides, iodides, or bromides,in the form of their hydroxides, carbonates, hydrogen carbonates, orsulfates, etc. Without being limited thereto, examples of sodium saltsinclude e.g. NaCl, NaI, NaBr, Na₂CO₃, NaHCO₃, Na₂SO₄, examples of theoptional potassium salts include e.g. KCl, KI, KBr, K₂CO₃, KHCO₃, K₂SO₄,and examples of calcium salts include e.g. CaCl₂, CaI₂, CaBr₂, CaCO₃,CaSO₄, Ca(OH)₂. Furthermore, organic anions of the aforementionedcations may be contained in the buffer. According to a more preferredaspect, the buffer suitable for injection purposes as defined above, maycontain salts selected from sodium chloride (NaCl), calcium chloride(CaCl₂) and optionally potassium chloride (KCl), wherein further anionsmay be present additional to the chlorides. CaCl₂ can also be replacedby another salt like KCl. Typically, the salts in the injection bufferare present in a concentration of at least 50 mM sodium chloride (NaCl),at least 3 mM potassium chloride (KCl) and at least 0.01 mM calciumchloride (CaCl₂). The injection buffer may be hypertonic, isotonic orhypotonic with reference to the specific reference medium, i.e. thebuffer may have a higher, identical or lower salt content with referenceto the specific reference medium, wherein preferably such concentrationsof the afore mentioned salts may be used, which do not lead to damage ofcells due to osmosis or other concentration effects. Reference media aree.g. liquids occurring in “in vivo” methods, such as blood, lymph,cytosolic liquids, or other body liquids, or e.g. liquids, which may beused as reference media in “in vitro” methods, such as common buffers orliquids. Such common buffers or liquids are known to a skilled personand may be as defined above.

However, one or more compatible solid or liquid fillers or diluents orencapsulating compounds may be used as well for the inventivepharmaceutical composition, which are suitable for administration to apatient to be treated. The term “compatible” as used here means thatthese constituents of the inventive pharmaceutical composition arecapable of being mixed with the dry powder composition or the particlesas defined herein in such a manner that no interaction occurs, whichwould substantially reduce the pharmaceutical effectiveness of theinventive pharmaceutical composition under typical use conditions.Pharmaceutically acceptable carriers, fillers and diluents must, ofcourse, have sufficiently high purity and sufficiently low toxicity tomake them suitable for administration to a person to be treated. Someexamples of compounds, which can be used as pharmaceutically acceptablecarriers, fillers or constituents thereof are sugars, such as, forexample, lactose, glucose and sucrose; starches, such as, for example,corn starch or potato starch; cellulose and its derivatives, such as,for example, sodium carboxymethylcellulose, ethylcellulose, celluloseacetate; powdered tragacanth; malt; gelatin; tallow; solid glidants,such as, for example, stearic acid, magnesium stearate; calcium sulfate;vegetable oils, such as, for example, groundnut oil, cottonseed oil,sesame oil, olive oil, corn oil and oil from theobroma; polyols, suchas, for example, polypropylene glycol, glycerol, sorbitol, mannitol andpolyethylene glycol; alginic acid.

The inventive pharmaceutical composition may be administered orally,parenterally, by inhalation, topically, rectally, nasally, buccally,vaginally or via an implanted reservoir. The term parenteral as usedherein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional, intracranial, transdermal, intradermal,intrapulmonal, intraperitoneal, intracardial, intraarterial, andsublingual injection or infusion techniques.

Preferably, the inventive pharmaceutical composition may be administeredby parenteral injection, more preferably by subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional, intracranial, transdermal,intradermal, intrapulmonal, intraperitoneal, intracardial,intraarterial, and sublingual injection or via infusion techniques.Sterile injectable forms of the inventive pharmaceutical compositionsmay be aqueous or oleaginous suspension. These suspensions may beformulated according to techniques known in the art using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersant,such as carboxymethyl cellulose or similar dispersing agents that arecommonly used in the formulation of pharmaceutically acceptable dosageforms including emulsions and suspensions. Other commonly usedsurfactants, such as Tweens, Spans and other emulsifying agents orbioavailability enhancers which are commonly used in the manufacture ofpharmaceutically acceptable solid, liquid, or other dosage forms mayalso be used for the purposes of formulation of the inventivepharmaceutical composition.

The inventive pharmaceutical composition as defined above may also beadministered orally in any orally acceptable dosage form including, butnot limited to, capsules, tablets, aqueous suspensions or solutions. Inthe case of tablets for oral use, carriers commonly used include lactoseand corn starch. Lubricating agents, such as magnesium stearate, arealso typically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient, i.e. the dry powdercomposition or the particles, as defined above, is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

The inventive pharmaceutical composition may also be administeredtopically, especially when the target of treatment includes areas ororgans readily accessible by topical application, e.g. includingdiseases of the skin or of any other accessible epithelial tissue.Suitable topical formulations are readily prepared for each of theseareas or organs. For topical applications, the inventive pharmaceuticalcomposition may be formulated in a suitable ointment, containing thecomponents as defined above suspended or dissolved in one or morecarriers. Carriers for topical administration include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the inventive pharmaceutical composition can beformulated in a suitable lotion or cream. In the context of the presentinvention, suitable carriers include, but are not limited to, mineraloil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearylalcohol, 2-octyldodecanol, benzyl alcohol and water.

In a particularly preferred embodiment, the inventive pharmaceuticalcomposition, preferably as an aerosolizable formulation, is for mucosal,intranasal, inhalation or pulmonary delivery. In a preferred embodiment,the pharmaceutical composition comprises the powder or the particles,preferably as defined herein, which are respirable, i.e. which canreadily be dispersed in air or in a gas (e.g. by using an inhalationdevice) and inhaled by a subject. Preferably, the particles of thepharmaceutical composition are as defined herein with respect to theinventive dry powder composition so that at least a portion of theaerosolized particles reaches the lungs. In a preferred embodiment, thepharmaceutical composition according to the invention comprises theinventive dry powder composition, wherein the dry powder compositioncomprises particles that have a MMAD of 10 μm or less.

The inventive pharmaceutical composition typically comprises a “safe andeffective amount” of the components of the inventive pharmaceuticalcomposition as defined above, particularly of long-chain RNA molecule ascomprised in the inventive dry powder composition or in the particlesobtainable by the inventive method. As used herein, a “safe andeffective amount” means an amount of the long-chain RNA molecule that issufficient to significantly induce a positive modification of a diseaseor disorder as defined herein. At the same time, however, a “safe andeffective amount” is small enough to avoid serious side-effects, that isto say to permit a sensible relationship between advantage and risk. Thedetermination of these limits typically lies within the scope ofsensible medical judgment. A “safe and effective amount” of thecomponents of the inventive pharmaceutical composition, particularly ofthe long-chain RNA molecule will furthermore vary in connection with theparticular condition to be treated and also with the age and physicalcondition of the patient to be treated, the body weight, general health,sex, diet, time of administration, rate of excretion, drug combination,the activity of the specific (lyophilized) nucleic acid (sequence)employed, the severity of the condition, the duration of the treatment,the nature of the accompanying therapy, of the particularpharmaceutically acceptable carrier used, and similar factors, withinthe knowledge and experience of the accompanying doctor. The inventivepharmaceutical composition may be used for human and also for veterinarymedical purposes, preferably for human medical purposes, as apharmaceutical composition in general or as a vaccine.

According to a specific aspect, the inventive dry powder composition,the particles or the dry powder composition obtainable by the inventivemethod or the inventive pharmaceutical composition may be provided as avaccine. Such an inventive vaccine is typically composed like theinventive pharmaceutical composition, i.e. it contains a long-chain RNAmolecule formulated as defined above and optionally a pharmaceuticallyacceptable carrier and/or vehicle. Further components may be as definedabove for the inventive pharmaceutical composition. The inventivevaccine preferably supports at least an innate immune response of theimmune system of a patient to be treated. Additionally, the inventivevaccine furthermore may also elicit an adaptive immune response,preferably, if the long-chain RNA molecule of the inventive vaccineencodes any of the antigens (or antibodies) mentioned herein, whichelicit an adaptive immune response or any antigen as defined herein isadded to the inventive vaccine, which can effectively induce an adaptiveimmune response.

The inventive vaccine may also comprise a pharmaceutically acceptablecarrier, adjuvant, and/or vehicle as defined above for the inventivepharmaceutical composition. In the specific context of the inventivevaccine, the choice of a pharmaceutically acceptable carrier isdetermined in principle by the manner in which the inventive vaccine isadministered. The inventive vaccine can be administered, for example,systemically or locally. Routes for systemic administration in generalinclude, for example, transdermal, oral, parenteral routes, includingsubcutaneous, intravenous, intramuscular, intraarterial, intradermal andintraperitoneal injections and/or intranasal/intrapulmonaladministration routes. Routes for local administration in generalinclude, for example, topical administration routes but alsointradermal, transdermal, subcutaneous, or intramuscular injections orintralesional, intracranial, intrapulmonal, intracardial, and sublingualinjections. More preferably, vaccines herein may be administered by anintradermal, subcutaneous, or intramuscular route. Inventive vaccinesare therefore preferably formulated in liquid (or sometimes in solid,e.g. as an aerosol) form. The suitable amount of the inventive vaccineto be administered can be determined by routine experiments with animalmodels. Such models include, without implying any limitation, rabbit,sheep, mouse, rat, dog and non-human primate models. Preferred unit doseforms for injection include sterile solutions of water, physiologicalsaline or mixtures thereof. The pH of such solutions should be adjustedto about 7.4. Suitable carriers for injection include hydrogels, devicesfor controlled or delayed release, polylactic acid and collagenmatrices. Suitable pharmaceutically acceptable carriers for topicalapplication include those, which are suitable for use in lotions,creams, gels and the like. If the inventive vaccine is to beadministered orally, tablets, capsules and the like are the preferredunit dose form. The pharmaceutically acceptable carriers for thepreparation of unit dose forms, which can be used for oraladministration, are well known in the prior art. The choice thereof willdepend on secondary considerations such as taste, costs and storability,which are not critical for the purposes of the present invention, andcan be made without difficulty by a person skilled in the art.

Further additives which may be included in the inventive vaccine areemulsifiers, such as, for example, Tween®; wetting agents, such as, forexample, sodium lauryl sulfate; colouring agents; taste-impartingagents, pharmaceutical carriers; tablet-forming agents; stabilizers;antioxidants; preservatives.

According to a specific embodiment, the inventive vaccine may comprisean adjuvant. In this context, an adjuvant may be understood as anycompound, which is suitable to initiate or increase an immune responseof the innate immune system, i.e. a non-specific immune response. Inother terms, when administered, the inventive vaccine preferably elicitsan innate immune response due to the adjuvant, optionally containedtherein. Preferably, such an adjuvant may be selected from an adjuvantknown to a skilled person and suitable for the present case, i.e.supporting the induction of an innate immune response in a mammal.

In this context, the adjuvant is preferably selected from compounds,which are known to be immune-stimulating due to their binding affinity(as ligands) to human Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5,TLR6, TLR7, TLR8, TLR9, TLR10, or due to its binding affinity (asligands) to murine Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5,TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13.

The present invention furthermore provides several applications and usesof the inventive dry powder composition, the particles obtainable by theinventive method or the dry powder composition obtainable by theinventive method. According to one aspect, the invention concerns theuse of the inventive dry powder composition, the particles obtainable bythe inventive method or the dry powder composition obtainable by theinventive method for the preparation of a medicament for theprophylaxis, treatment and/or amelioration of a disorder or a disease,preferably as defined herein.

According to a further aspect, the present invention is directed to theuse of the long-chain RNA molecule in particulate form as defined hereinin the treatment or prevention of a disease. Further, the inventionconcerns the use of the inventive dry powder composition, the particlesobtainable by the inventive method or the dry powder compositionobtainable by the inventive method, in the treatment or the preventionof a disease, preferably as defined herein. In particular, the presentinvention concerns the first medical use of the inventive dry powdercomposition, the particles obtainable by the inventive method or the drypowder composition obtainable by the inventive method as a medicament.The medicament may be in the form of a pharmaceutical composition or inthe form of a vaccine as a specific form of pharmaceutical compositions.A pharmaceutical composition in the context of the present inventiontypically comprises or consists of the inventive dry powder composition,the particles obtainable by the inventive method or the dry powdercomposition obtainable by the inventive method as defined above,optionally further ingredients, preferably as defined above, andoptionally a pharmaceutically acceptable carrier and/or vehicle,preferably as defined above.

According to a further aspect, the present invention concerns a methodof treating or preventing a disorder or a disease by administering to asubject in need thereof a pharmaceutically effective amount, preferablyas defined herein, of the inventive dry powder composition, theinventive pharmaceutical composition, or the inventive vaccine.Preferably, the method is for treating or preventing a disorder or adisease selected from cancer or tumor diseases, infectious diseases,preferably (viral, bacterial or protozoological) infectious diseases,autoimmune diseases, allergies or allergic diseases, monogeneticdiseases, i.e. (hereditary) diseases, or genetic diseases in general,diseases which have a genetic inherited background and which aretypically caused by a single gene defect and are inherited according toMendel's laws, cardiovascular diseases, neuronal diseases, or anyfurther disease mentioned herein.

According to one further aspect, the present invention is directed tothe use of the long-chain RNA molecule in particulate form, preferablyin the form of the inventive dry powder composition, the particlesobtainable by the inventive method or the dry powder compositionobtainable by the inventive method, for the prophylaxis, treatmentand/or amelioration of a disease or disorder as defined herein, whereinthe disease or disorder is preferably selected from cancer or tumordiseases, infectious diseases, preferably (viral, bacterial orprotozoological) infectious diseases, autoimmune diseases, allergies orallergic diseases, monogenetic diseases, i.e. (hereditary) diseases, orgenetic diseases in general, diseases which have a genetic inheritedbackground and which are typically caused by a single gene defect andare inherited according to Mendel's laws, cardiovascular diseases,neuronal diseases, or any further disease mentioned herein.

According to another aspect, the present invention is directed to thesecond medical use of the long-chain RNA in particulate form as definedherein, preferably in the form of the inventive dry powder composition,the particles obtainable by the inventive method or the dry powdercomposition obtainable by the inventive method for the treatment ofdiseases as defined herein, preferably to the use thereof for thepreparation of a medicament for the prophylaxis, treatment and/oramelioration of various diseases as defined herein, preferably selectedfrom cancer or tumor diseases, infectious diseases, preferably (viral,bacterial or protozoological) infectious diseases, autoimmune diseases,allergies or allergic diseases, monogenetic diseases, i.e. (hereditary)diseases, or genetic diseases in general, diseases which have a geneticinherited background and which are typically caused by a single genedefect and are inherited according to Mendel's laws, cardiovasculardiseases, neuronal diseases, or any further disease mentioned herein.

The present invention also allows treatment of diseases, which have notbeen inherited, or which may not be summarized under the abovecategories. Such diseases may include e.g. the treatment of patients,which are in need of a specific protein factor, e.g. a specifictherapeutically active protein as mentioned above. This may e.g. includedialysis patients, e.g. patients, which undergo a (regular) a kidney orrenal dialysis, and which may be in need of specific therapeuticallyactive proteins as defined above, e.g. erythropoietin (EPO), etc.

Likewise, diseases in the context of the present invention may includecardiovascular diseases chosen from, without being limited thereto,coronary heart disease, arteriosclerosis, apoplexy and hypertension,etc.

Finally, diseases in the context of the present invention may be chosenfrom neuronal diseases including e.g. Alzheimer's disease, amyotrophiclateral sclerosis, dystonia, epilepsy, multiple sclerosis andParkinson's disease etc.

According to a further embodiment, the present invention also provides akit, particularly as a kit of parts. Such a kit of parts may containe.g. the inventive dry powder composition, the inventive pharmaceuticalcomposition or the inventive vaccine as defined above, preferablydivided into different parts of the kit. As an example, the inventivepharmaceutical composition or the inventive vaccine may be prepared as akit of parts, e.g. by incorporating into one or more parts of the kit(all or at least some components of) the inventive pharmaceuticalcomposition or the inventive vaccine as described herein (whereby atleast the long-chain RNA in particulate form is included), or theinventive dry powder composition as such, as a dry formulation, i.e.devoid of any liquid component, and in at least one further separatepart of the kit a solvent and/or a buffer as described herein withrespect to the liquid provided in step a) of the inventive method, theinventive pharmaceutical composition or the inventive vaccine or anyfurther solvent and/or buffer as described herein for lyophilization,transfection and/or injection. Alternatively, the inventivepharmaceutical composition or the inventive vaccine may be prepared as akit of parts, e.g. by incorporating into one or more parts of the kitonly the inventive dry powder composition, the particles obtainable bythe inventive method, or the dry powder composition obtainable by theinventive method, as described herein, and in at least one furtherseparate part of the kit a solvent and/or a buffer as described hereinfor the liquid provided in step a) of the inventive method, for theinventive pharmaceutical composition or the inventive vaccine or anyfurther liquid and/or buffer as described herein for lyophilization,transfection and/or injection. Without being limited thereto, furtheringredients of the kit may include components as defined above, e.g.(solutions comprising) proteins, amino acids, alcohols, carbohydrates,metals or metal ions, surfactants, polymers or complexing agents, and/orbuffers, preferably all as defined above. These further ingredients maybe contained in different parts of the kit (or kit of parts). The kit orkit of parts as described above may contain optionally technicalinstructions with information on the administration and dosage of theinventive composition. Such a kit, preferably kit of parts, may beapplied, e.g., for any of the above mentioned applications or uses.

BRIEF DESCRIPTION OF THE FIGURES

The figures shown in the following are merely illustrative and shalldescribe the present invention in a further way. These figures shall notbe construed to limit the present invention thereto.

FIG. 1: Scheme of a co-current spray-drying apparatus

-   -   A: solution or suspension to be dried inlet, B: atomization gas        (e.g. nitrogen) inlet, 1: drying gas (e.g. nitrogen) inlet, 2:        heating of drying gas, 3: spraying of solution or suspension, 4:        drying chamber, 5: part between drying chamber and cyclone, 6:        cyclone, 7: drying gas outlet, 8: product collection vessel.

FIG. 2: Sequence of the mRNA used in this study (R2564; SEQ ID NO: 1).

FIG. 3: Photograph of powder of protamine-formulated RNA (T-SD1, T-SD2,T-SD3) spray-dried with outlet temperatures of 47, 69 and 87° C.respectively and of placebo sample (T-SD-P) spray-dried at 84° C.

FIG. 4: Residual water content of dry powder formulations obtained byspray-drying using different outlet temperatures.

FIG. 5: X-ray powder diffraction analysis of protamine-formulated RNA(T-SD1, T-SD2, T-SD3) spray-dried with outlet temperatures of 47, 69 and87° C. respectively and placebo sample (T-SD-P) spray-dried at 84° C.

FIG. 6: Particle size distributions of protamine-formulated RNA (T-SD1,T-SD2, T-SD3) spray-dried with outlet temperatures of 47, 69 and 87° C.respectively and placebo sample (T-SD-P) spray-dried at 84° C. asdetermined by laser diffraction analysis.

FIG. 7: Scanning electron microscope (SEM) images ofprotamine-formulated RNA powder particles (T-SD-1 (FIG. 7A, 5100×),T-SD-2 (FIG. 7B, 4950×), T-SD-3 (FIG. 7C, 2080×) and placebo powderparticles (T-SD-P, FIG. 7D, 2660×).

FIG. 8: Particle size distribution of protamine-formulated RNA before(T0) and after (T-SD 1, T-SD 2, T-SD 3) spray-drying with outlettemperatures of 47, 69 and 87° C. respectively and particle size ofspray-dried placebo sample (T-SD-P) with an outlet temperature of 84° C.The particle size was determined by dynamic light scattering (DLS).

FIG. 9: Particle size distribution of protamine-formulated RNA before(T0) and after (T-SD 1, T-SD 2, T-SD 3) spray-drying with outlettemperatures of 47, 69 and 87° C. respectively and particle size ofspray-dried placebo sample (T-SD-P) with an outlet temperature of 84° C.The particle size was determined by nanoparticle tracking analysis(NTA).

EXAMPLES

The Examples shown in the following are merely illustrative and shalldescribe the present invention in a further way. These Examples shallnot be construed to limit the present invention thereto.

Example 1: Preparation of DNA and RNA Constructs

A vector for in vitro transcription was constructed containing a T7promoter followed by a GC-enriched sequence encoding the hemagglutinin(HA) protein of influenza A virus (A/Netherlands/602/2009(H1N1)) andused for subsequent in vitro transcription reactions. According to afirst preparation, the DNA sequence coding for the above mentioned mRNAwas prepared. The constructs R2564 (SEQ ID NO: 1) was prepared byintroducing a 5′-TOP-UTR derived from the ribosomal protein 32L4,modifying the wild type coding sequence by introducing a GC-optimizedsequence for stabilization, followed by a stabilizing sequence derivedfrom the albumin-3′-UTR, a stretch of 64 adenosines (poly(A)-sequence),a stretch of 30 cytosines (poly(C)-sequence), and a histone stem loop.In SEQ ID NO: 1 (see FIG. 2) and the sequence of the corresponding mRNAis shown.

Example 2: In Vitro Transcription and Purification of RNA

The respective DNA plasmids prepared according to section 1 above weretranscribed in vitro using T7 polymerase. The in vitro transcription ofinfluenza HA encoding R2564 was performed in the presence of a CAPanalog (m7GpppG). Subsequently the RNA was purified using PureMessenger®(CureVac, Tübingen, Germany; WO2008/077592A1).

Example 3: Preparation of Protamine-Formulated RNA

RNA was diluted (0.87 g/L RNA final concentration) and aprotamine/trehalose mixture was prepared (43000 anti-heparin IU/Lprotamine; 10.87% trehalose in water for injection). One volume unit ofeach solution was mixed to yield a ratio of protamine to RNA of 50anti-heparin IU per mg RNA.

The solution of RNA/protamine complexes were supplemented with R2564 toyield final concentrations of 0.4 g/L RNA complexed with 20000anti-heparin IU/L of protamine (corresponding to a protamineconcentration of about 1.5 g/L), 0.4 g/L free RNA and 5% trehalose(w/w).

Such formulated RNA was used for spray-drying experiments.

As a placebo, 5% trehalose was prepared in water for injection.

Example 4: Spray-Drying of Protamine-Formulated RNA and PlaceboFormulation

The objective of the experiments presented in this section was to testthe feasibility of large scale production of the inventive dry powdercomposition by spray-drying. In summary, three spray-drying experimentswere performed at different outlet temperatures using theprotamine-formulated RNA prepared according to Example 3. As a control,the placebo formulation as described in Example 3 was processed inparallel.

Protamine-formulated RNA (Example 3) or placebo sample was thawed andeach aliquot was homogenized by gentle mixing using a magnetic stirrerbefore spray-drying.

Spray-drying of protamine-formulated RNA and placebo formulation wascarried out using a Büchi Mini Spray-Dryer B-290 equipped with atwo-fluid nozzle and a high performance cyclone. The spray-dryer wasoperated using nitrogen as drying gas in a closed cycle mode. Thespray-drying experiments were carried out under the process parameterslisted in Table 1.

TABLE 1 Process parameters spray-drying Process T-SD1 T-SD2 T-SD3parameter (verum) (verum) (verum) T-SP-P Nozzle type two-fluid two-fluidtwo-fluid two-fluid nozzle nozzle nozzle nozzle Atomization 30 mm ± 30mm ± 30 mm ± 30 mm ± gas flow 5 mm 5 mm 5 mm 5 mm setting* (~357 l/h)(~357 l/h) (~357 l/h) (~357 l/h) (theoretical volume flow) Inlet 65° C.100° C. 128° C. 128° C. temperature Outlet 47° C.  69° C.  87° C.  84°C. temperature Drying gas nitrogen nitrogen nitrogen nitrogen Drying gas100% 100% 100% 100% rate/aspirator (~35 m³/h) (~35 m³/h) (~35 m³/h) (~35m³/h) Pump speed 3% 3% 3% 3% (~1 ml/min) (~1 ml/min) (~1 ml/min) (~1ml/min) Yield [g] 0.788 0.915 1.017 0.909* Calculated 44.2 51.4 56.548.3* relative yield [%] *Atomization gas flow setting of 30 mmcorrelates with a pressure drop at the nozzle of 0.23 bar

Following spray-drying, the produced powders were filled into vials (seesection 5) and characterized (see section 6).

5. Powder Filling

The powder obtained after spray-drying was collected in a glasscontainer at the product outlet of the cyclone. For storage, shipmentand further analysis the powder was divided into aliquots andtransferred into 10R vials under controlled humidity conditions (<15%RH) in a glove box. Vials were stoppered inside the glove box. The exactweight of the dry powder was documented during filling. FIG. 3 shows aphotograph of glass vials containing the spray-dried powder.

6. Analytical Characterization

A sampling scheme, including the sampling time points and analyticalmethods is shown in Table 2.

TABLE 2 sampling time points for samples from spray-drying Time pointDescription analytical methods T0 (verum) Liquid verum sample DLS, NTA,MFI, turbidity, ZP before spray-drying. T0-P Liquid placebo DLS, NTA,MFI, turbidity formulation before spray-drying T-SDX Spray-dried verumDLS, ZP, NTA, MFI, turbidity sample obtained from Karl-Fischertitration, experiment T-SD1, DSC, XRD, laser T-SD2, T-SD3, T-SD4diffraction analysis, SEM T-SD-P Spray-dried placebo DLS, NTA, MFI,turbidity sample obtained from experiment T-SD-P

6.1 Methods for Physico-Chemical Characterization of Spray-Dried Powder

Spray-dried powders were characterized with respect to physico-chemicalproperties of the spray-dried formulation using various methods (Table3).

TABLE 3 Analytical methods for physico-chemical characterization ofspray-dried powder. Sample Abbreviation Full term Dry powder DSCDifferential scanning calorimetry KF Karl Fischer titration XRD X-raypowder diffraction Laser diffraction Laser diffraction analysis SEMScanning electron microscopy

6.1.1 Karl Fischer Titration

The residual moisture content of the dried powders were determined usingthe coulometric Karl Fischer titrator Aqua 40.00 (Analytik Jena GmbH,Jena, Germany), which is equipped with a headspace module.

As a system suitability check, the residual moisture content of a PureWater Standard (Apura 1 water standard oven 1.0, Merck KGaA) wasanalyzed prior to sample measurement. The residual moisture content ofthe standard had to be within 1.00±0.03% in order to comply with themanufacturer specifications.

For the measurement, about 20 mg of sample were weighed into 2R glassvials and heated to a measurement temperature of 120° C. in the ovenconnected to the reaction vessel via a tubing system. The evaporatedwater was transferred into the titration solution and the amount ofwater was determined. The measurement was performed until no more waterevaporation was detectable (actual drift comparable to drift at thebeginning of the measurement). Ambient moisture was determined bymeasurement of three blanks (empty vials prepared in the preparationenvironment). Results obtained for samples were corrected for thedetermined ambient moisture by blank subtraction. Samples were measuredin duplicates. The results are shown in FIG. 4 and Table 4.

TABLE 4 Water content of spray-dried formulation Sample Water content[%] T-SD1 4.37 T-SD2 2.81 T-SD3 1.78 T-SD-P 1.70

6.1.2 Differential Scanning Calorimetry (DSC)

Differential scanning calorimetry (DSC) in a Mettler Toledo 821e(Mettler Toledo, Giessen, Germany) was used to determine thermal eventsof the spray-dried samples (e.g. glass transition temperature (Tg),crystallization or endothermal melting). Approximately 10 mg of thespray-dried samples were analyzed in crimped Al-crucibles (MettlerToledo, Giessen, Germany). The samples were cooled to 0° C. at a coolingrate of 10 K/min and reheated to 120° C. with a rate of 10 K/min. Themeasurement of the temperature profile was repeated in a second cycle inorder to evaluate reversibility of thermal events. The Tg was determinedas the midpoint of the endothermic shift of the baseline during theheating scan (see Table 5). The maximum of exothermic/endothermic peakswere reported as Tcryst/Tm.

TABLE 5 Tg values of spray-dried formulation Sample Tg (1^(st) scan) [°C.] T-SD1 59.0 T-SD2 72.1 T-SD3 87.9 T-SD-P 87.8

Tg values correlate with the water contents of the samples. Norelaxation phenomenon (exothermic event) was detectable in spray-driedsamples, in contrast to thermograms obtained for lyophilized samples.

6.1.3 X-Ray Powder Diffraction (XRD)

Wide angle X-ray powder diffraction (XRD) was used to study themorphology of lyophilized products. The X-ray diffractometer Empyrean(Panalytical, Almelo, The Netherlands) equipped with a copper anode (45kV, 40 mA, Kα1 emission at a wavelength of 0.154 nm) and a PIXcel3Ddetector was used. Approximately 100 mg of the spray-dried samples wereanalyzed in reflection mode in the angular range from 5-45° 2θ, with astep size of 0.04° 2θ and a counting time of 100 seconds per step. Therespective XRD diagrams are shown in FIG. 5.

All samples showed an amorphous pattern and no indication of crystallinephases.

6.1.4 Laser Diffraction Analysis

Size distribution of spray-dried powders were measured by laserdiffraction. Laser diffraction measurements were performed using aPartica LA-950 Laser Diffraction Particle Size Distribution Analyzer(Horiba, Kyoto, Japan) equipped with a 605 nm laser diode for detectingparticles>500 nm and 405 nm blue light emitting diode (LED) fordetecting particles<500 nm. The powder samples were dispersed in Miglyol812 by ultra sonication for up to 5 min. Prior to measurement, thesystem was blanked with Miglyol 812. Each sample dispersion was measured3 times. Measurement results were analyzed using Horiba LA-950 Software.

The results were reported as

D10: particle diameter corresponding to 10% of the cumulative undersizedistribution;D50: particle diameter corresponding to 50% of the cumulative undersizedistribution;D90: particle diameter corresponding to 90% of the cumulative undersizedistribution.

The results are summarized in Table 6 and FIG. 6.

TABLE 6 Particle size distribution of spray-dried powders as measured bylaser diffraction Median Modal Mean Sample Absorbance Diameter DiameterValue St. Dev. D10 D50 D90 T-SDP 0.126 4.21 6.75 4.032 0.394 1.204 4.2112.516 T-SD3 0.217 4.132 6.75 3.615 0.408 0.938 4.132 11.151 T-SD2 0.1874.31 6.75 3.728 0.418 0.933 4.31 11.588 T-SD1 0.051 4.608 6.75 3.9270.375 1.075 4.608 10.854 (sizes are indicated in μm)

6.1.5 Scanning Electron Microscopy (SEM)

Images of spray-dried powder particles were generated by using the benchtop scanning electron microscope Phenom (Phenom-World B.V., Eindhoven,Netherlands). The instrument is equipped with a CCD camera and adiaphragm vacuum pump. Each sample was prepared in a glove box undercontrolled humidity conditions (<20% relative humidity) by using thefollowing method: a small amount of the powder was carefully put on aself-adhesive carbon foil placed on a sample holder. The sample wasanalyzed under vacuum with a light optical magnification of 24× and 5 kVacceleration voltage. The electron optical magnification was adjustedbetween 1160× and 1700× and images were made from representativesections of each sample.

The obtained images (see FIG. 7) demonstrate that the obtained powderparticles have spherical shape and that the size of the powder particlesis in the range from less than 1 μm to approximately 20 μm.

6.2 Reconstitution of Spray-Dried Samples

For reconstitution of the spray-dried samples, the reconstitution volumewas calculated for each sample individually based on the amount ofpowder weighed into the vial. The calculation was based on the methodfor reconstitution of lyophilized samples (addition of 600 μl water forinjection to 30.6 mg powder per vial).

The reconstitution volume for varying amounts of spray-dried powder wascalculated according to the following equation:

V _(reconst.) =m _(powder)*1000 μl/51 mg

V_(reconst.): reconstitution volume in mlm_(powder): mass of powder to be reconstituted in mg(based on a theoretical solid content of 51 mg per ml (50 mg/mltrehalose, 0.8 mg/ml RNA (free+complexed), 20 anti-heparin IU/mLprotamine))

The spray-dried samples were reconstituted under laminar flow conditionsusing a procedure comparable to the procedure for the lyophilizedproduct: cap and stopper were removed from the vial and the calculatedvolume of water for injection was added to the dry powder (into thecenter of the vial) by using a multipette with 10 ml combitip. The vialwas carefully slewed (shaking was avoided), until all powder particleswere dissolved. The reconstitution time was measured as the timerequired in order to achieve full reconstitution of the dry powder afterthe liquid has been added. The reconstitution behavior was judged,mainly with respect to foaming, and recorded (see Table 7).

TABLE 7 Reconstitution behaviour Sample Reconstitution time [mm:ss] Foamformation T-SD1 01:27 0 T-SD2 01:20 0 T-SD3 01:37 0 T-SD-P 01:04 0

In conclusion, reconstitution times for the spray-dried formulationswere below 2 minutes. A slightly shorter resolution time was observedfor the placebo formulation (T-SD-P).

6.3 Particle Characterization

The particles comprised in liquid samples of the protamine-formulatedRNA as described above were characterized before spray-drying, and afterreconstitution of the spray-dried samples (Table 8).

TABLE 8 Analytical methods for characterization of liquid samples(before and after spray- drying and reconstitution). Sample AbbreviationFull term Liquid sample Visual Visual inspection DLS Dynamic lightscattering MFI Micro-Flow Imaging Turbidity Turbidity NTA Nanoparticletracking analysis ZP Zeta potential

6.3.1 Visual Inspection

For visual inspection, the reconstituted vials were inspected for thepresence or absence of visible particles under gentle, manual, radialagitation for 5 sec in front of a white background and for 5 sec infront of a black background according to the European Pharmacopoeia. Theinspection was performed by two independent examiners. To furtherclassify the particle content, the method described in the “DeutscherArzneimittel-Codex” (DAC) was used.

The classification can be described as follows:

No particles visible within 5 sec: 0 pointFew particles visible within 5 sec: 1 pointMedium number of particles visible within 5 sec: 2 pointsLarge number of particles directly visible: 10 points(Particles that were on the limit of being visible as distinct particles(cloudiness, schlieren) were rated with 2 points.)

The results of the visual inspection of the liquid samples are recordedin Table 9.

TABLE 9 Results of visual inspection Sample Score [Exp 1/2] T-SD1 2*/2*T-SD2 1*/2  T-SD3 1/1 T-SD-P 2*/2* *fiber-like particle(s)

As a result, visible particles were observed in all of the analyzedsamples. The majority of the observed particles were fiber-likeparticles that were likely due to a contamination.

6.3.2 Turbidity

The NEPHLA turbidimeter (Dr. Lange, Düsseldorf, Germany), operating at860 nm and detecting at 90° angle, was used for turbidity measurements.The system was calibrated with formazin as a standard and the resultswere given in formazin nephelometric units (FNU).

For the measurement, 2.0 ml solution were analyzed in a clean glasscuvette. The turbidity of the individual samples is indicated in Table10. After analysis, the sample material was used for further analysis(e.g. DLS, MFI, etc.).

TABLE 10 Turbidity of the liquid samples Sample Turbidity [FNU] T0 20.1T-SD1 26.1 T-SD2 17.7 T-SD3 18.0 T-SD-P 3.5

In summary, the turbidity of the spray-dried protamine-formulated RNAafter reconstitution varied from 18 to 26 FNU.

6.3.3 Dynamic Light Scattering (DLS)

DLS measurements were carried out by using a Zetasizer Nano Series(Malvern Instruments, Worcestershire, UK) instrument. 150 μl of thesample were analyzed in small volume disposable cuvettes (UVette) byusing an automated mode for each sample. As a control (T0), theprotamine-formulated RNA before spray-drying was used.

The Malvern Zetasizer Software was used to calculate Z-average diameter,polydispersity index (PDI) and an intensity size distribution(refractive index and viscosity of water was selected in the software).The results are shown in Table 11 and FIG. 8.

TABLE 11 Z-average diameter, polydispersity index, main peak diameterand main peak intensity as determined by Dynamic light scatteringZ-average Main peak Main peak diameter diameter intensity Derived CountSample [nm] PdI [nm] [%] Rate T0 218.7 ± 0.5 0.183 ± 0.012 263.1 ± 3.6100 ± 0 55383 ± 219 T-SD1 282.9 ± 8.7 0.128 ± 0.103  325.6 ± 19.4 100 ±0 54725 ± 363 T-SD2 267.3 ± 4.9 0.208 ± 0.021 328.3 ± 4.6 100 ± 0 48428± 394 T-SD3 292.7 ± 4.9 0.252 ± 0.012 380.4 ± 3.6 100 ± 0 39503 ± 42 T-SD-P  425.2 ± 36.5 0.418 ± 0.050  252.3 ± 15.3  98.3 ± 3.0  1989 ± 197

As a result, slightly increased Z-average and main peak diameters weredetermined for spray dried samples.

6.3.4 Nanoparticle Tracking Analysis (NTA)

NTA experiments were carried out with a NanoSight LM20 (NanoSight,Amesbury, UK). The instrument is equipped with a 405 nm blue laser, asample chamber and a Viton fluoroelastomer O-Ring. The samples werediluted with ultra-pure water in order to achieve suitableconcentrations for NTA measurement. After the measurement, all resultswere normalized to the original concentration.

Samples were loaded into the measurement cell using a 1 ml syringe. Theresults of the NTA analysis are shown in Table 12 and FIG. 11. Movementsof the particles in the samples were recorded as videos for 60 secondsat room temperature using the NTA 2.0 Software. The recorded videos wereanalyzed with the NTA 2.0 Software.

TABLE 12 Results from NTA analysis Mean size Mode size D10 size D50 sizeD90 size Total Concentration Sample [nm] [nm] [nm] [nm] [nm] [#/ml] T0108 ± 2 96 ± 7 71 ± 1 102 ± 3 150 ± 5 4.74 (±0.16) E+11 T-SD1 109 ± 6 92± 9 73 ± 5 102 ± 9 149 ± 5 3.96 (±0.89) E+11 T-SD2 120 ± 3 107 ± 6  83 ±3 114 ± 5 161 ± 2 5.71 (±0.42) E+11 T-SD3 136 ± 5 116 ± 10 86 ± 4 126 ±3  197 ± 13 6.04 (±0.59) E+11 T-SD-P  139 ± 30 131 ± 40 117 ± 41  136 ±33  161 ± 19 8.31 (±9.65) E+09

The particle size values determined for the spray-dried samples werecomparable or slightly increased with respect to the control (T0).

6.3.5 Zeta Potential Measurements

Zeta potential measurements were carried out with a Zetasizer NanoSeries instrument (Malvern Instruments, Worcestershire, UK). 750 μl ofeach formulation were analyzed in disposable folded capillary cells. Foreach sample, 3 zeta potential measurements consisting of 100 sub-runswere performed and the mean value for zeta potential was calculated. Forall samples, a negative zeta potential was determined (see Table 13).

TABLE 13 Results of Zeta potential measurements Sample Zeta potential[mV] T0 −32.4 T-SD1 −35.8 T-SD2 −36.7 T-SD3 −41.2 T-SD-P −22.5

6.3.6 Micro-Flow Imaging (MFI)

Micro-Flow Imaging measurements were conducted by using a DPA-5200particle analyzer system (ProteinSimple, Santa Clara, Calif., USA)equipped with a silane coated high resolution 100 μm flow cell. Sampleswere analyzed undiluted. In case of excess of the MFI concentrationslimits 2.5 μm: 900,000 particles/ml, 5 μm: 400,000 particles/ml and 10μm: 250,000 particles/mil), samples were diluted before analysis byadding ultrapure water (e.g. Milli-Q water).

For the analysis of the liquid samples, a pre-run volume of 0.17 ml wasfollowed by a sample run of 0.26 ml. Approximately 1100 images weretaken per sample. Between measurements, the flow cell was cleaned withwater and the background illumination was optimized by using ultrapurewater. The MFI View System Software (MVSS) version 2-R2-6.1.20.1915 wasused to perform the measurements and the MFI View Analysis Suite (MVAS)software version 1.3.0.1007 was used to analyze the data. The particlecounts of the diluted samples were normalized to the originalconcentration. Significantly increased particle concentrations weredetermined for the spray-dried samples including placebo (see Table 14),pointing towards particle contamination.

TABLE 14 Particle concentration as determined by MFI Particleconcentration [#/ml] Sample ≥1 μm ≥2 μm ≥10 μm ≥25 μm T0 12,509 4,115203 11 T-SD1 174,199 32,426 218 4 T-SD2 128,351 27,151 289 18 T-SD3139,397 28,440 225 7 T-SD-P 183,275 35,053 86 0

1. Dry powder composition comprising a long-chain RNA molecule.
 2. Thedry powder composition according to claim 1, which comprises a pluralityof particles.
 3. The dry powder composition according to claim 1 or 2having a residual moisture content of 7% (w/w) or less.
 4. The drypowder composition according to any one of claims 1 to 3, wherein themedian particle size in a volume weighted distribution is at least 1 μm.5. The dry powder composition according to any one of claims 1 to 4,wherein the average sphericity of the particles is in a range from 0.7to 1.0.
 6. The dry powder composition according to any one of claims 1to 5, wherein the long-chain RNA molecule is present in the form of afree long-chain RNA molecule, or in the form of a complex comprising thelong-chain RNA molecule.
 7. The dry powder composition according to anyone of claims 1 to 6, wherein the long-chain RNA molecule is in acomplex with a cationic or polycationic compound.
 8. The dry powdercomposition according to any one of claims 1 to 7, wherein thelong-chain RNA molecule is a single-stranded RNA molecule.
 9. The drypowder composition according to any one of claims 1 to 8, wherein thelong-chain RNA molecule comprises at least 30 nucleotides.
 10. The drypowder composition according to any one of claims 1 to 9, wherein thelong-chain RNA molecule comprises more than 200 nucleotides, preferablyat least 250 nucleotides.
 11. The dry powder composition according toany one of claims 1 to 10, wherein the long-chain RNA molecule comprisesat least one open reading frame (ORF) encoding a protein or a peptide.12. The dry powder composition according to any one of claims 1 to 11,wherein the long-chain RNA molecule is an mRNA molecule.
 13. The drypowder composition according to any one of claims 1 to 12, wherein thelong-chain RNA molecule comprises at least one modification.
 14. The drypowder composition according to any one of claims 1 to 13, whichcomprises at least one further excipient.
 15. Method for preparing a drypowder comprising a long-chain RNA molecule, wherein the methodcomprises the following steps: a) providing a liquid comprising thelong-chain RNA molecule, b) drying the liquid provided in step a) byspray-drying.
 16. The method according to claim 15, wherein thelong-chain RNA molecule is characterized by any one of the featuresdefined in any one of claims 6 to
 13. 17. The method according to claim15 or 16, wherein the liquid comprising the long-chain RNA moleculefurther comprises at least one excipient selected from a cryoprotectant,a lyoprotectant and a bulking agent.
 18. The method according to any oneof claims 15 to 17, wherein the liquid comprising the long-chain RNAmolecule does not contain a lipid compound.
 19. The method according toany one of claims 15 to 18, wherein the liquid comprising the long-chainRNA molecule comprises a solvent suitable for spray-drying.
 20. Themethod according to any one of claims 15 to 19, wherein T_(inlet) is atleast 85° C.
 21. The method according to any one of claims 15 to 20,wherein T_(outlet) is at least 50° C.
 22. The method according to anyone of claims 15 to 21, wherein the liquid comprising the long-chain RNAmolecule is atomized and the droplets resulting from the atomization ofthe liquid are characterized by a mass median aerodynamic diameter of300 nm to 200 μm.
 23. The method according to any one of claims 15 to22, wherein the liquid comprising the long-chain RNA molecule isatomized by using an atomizer selected from the group of rotaryatomizers, pressure nozzles, two-fluid nozzles, ultrasonic nebulizersand vibrating orifice aerosol generators.
 24. Dry powder compositionobtainable by the method according to any one of claims 15 to
 23. 25.Pharmaceutical composition comprising or consisting of the dry powdercomposition according to any one of claims 1 to 14 or the dry powdercomposition according to claim
 24. 26. The pharmaceutical compositionaccording to claim 25, which comprises at least one furtherpharmaceutically acceptable excipient.
 27. Vaccine comprising orconsisting of the dry powder composition according to any one of claims1 to 14 or the dry powder composition according to claim
 24. 28. Thevaccine according to claim 27, which comprises at least one furtherpharmaceutically acceptable excipient.
 29. The vaccine according toclaim 27 or 28, wherein the dry powder composition was reconstituted ina suitable solvent or buffer.
 30. Kit comprising the dry powdercomposition according to any one of claims 1 to 14 or 24, thepharmaceutical composition according to claim 25 or 26, or the vaccineaccording to any one of claims 27 to 29, a solvent or buffer forresuspending the dry powder composition, the pharmaceutical compositionor the vaccine, and optionally technical instructions comprisinginformation regarding the administration and/or dosage of the dry powdercomposition, the pharmaceutical composition or the vaccine.
 31. Kit ofparts comprising in one or more parts of kit the dry powder compositionaccording to any one of claims 1 to 14 or 24, the pharmaceuticalcomposition according to claim 25 or 26, or the vaccine according to anyone of claims 27 to 29, a solvent or buffer for resuspending the drypowder composition, the pharmaceutical composition or the vaccine, andoptionally technical instructions comprising information regarding theadministration and/or dosage of the dry powder composition, thepharmaceutical composition or the vaccine.
 32. Use of the dry powdercomposition according to any one of claims 1 to 14 or 24 for thepreparation of a medicament for the prophylaxis, treatment and/oramelioration of a disorder or a disease.
 33. The use according to claim32, wherein the disorder or disease is selected from the groupconsisting of cancer or tumor diseases, infectious diseases, preferablyviral, bacterial or protozoological infectious diseases, autoimmunediseases, allergies or allergic diseases, monogenetic diseases, i.e.(hereditary) diseases, or genetic diseases in general, diseases whichhave a genetic inherited background and which are typically caused by asingle gene defect and are inherited according to Mendel's laws,cardiovascular diseases and neuronal diseases.
 34. Method of treating orpreventing a disorder or a disease by administering to a subject in needthereof a pharmaceutically effective amount of the dry powdercomposition according to any one of claims 1 to 14 or 24, thepharmaceutical composition according to claim 25 or 26, or the vaccineaccording to any one of claims 27 to
 29. 35. The method according toclaim 34, wherein the disorder or disease is selected from the groupconsisting of cancer or tumor diseases, infectious diseases, preferablyviral, bacterial or protozoological infectious diseases, autoimmunediseases, allergies or allergic diseases, monogenetic diseases, i.e.(hereditary) diseases, or genetic diseases in general, diseases whichhave a genetic inherited background and which are typically caused by asingle gene defect and are inherited according to Mendel's laws,cardiovascular diseases and neuronal diseases.
 36. The dry powdercomposition according to any one of claims 1 to 14 or 24, thepharmaceutical composition according to claim 25 or 26, the vaccineaccording to any one of claims 27 to 29, the kit according to claim 30or the kit of parts according to claim 31, for use in the prophylaxis,treatment and/or amelioration of a disorder or disease.
 37. The drypowder composition according to any one of claims 1 to 14 or 24, thepharmaceutical composition according to claim 25 or 26, the vaccineaccording to any one of claims 27 to 29, the kit according to claim 30or the kit of parts according to claim 31, for use according to claim36, wherein the disorder or disease is selected from the groupconsisting of cancer or tumor diseases, infectious diseases, preferablyviral, bacterial or protozoological infectious diseases, autoimmunediseases, allergies or allergic diseases, monogenetic diseases, i.e.(hereditary) diseases, or genetic diseases in general, diseases whichhave a genetic inherited background and which are typically caused by asingle gene defect and are inherited according to Mendel's laws,cardiovascular diseases and neuronal diseases.